IgG Fc fragment for a drug carrier and method for the preparation thereof

ABSTRACT

Disclosed is an IgG Fc fragment useful as a drug carrier. A recombinant vector expressing the IgG Fc fragment, a transformant transformed with the recombinant vector, and a method of preparing an IgG Fc fragment are disclosed. When conjugated to a certain drug, the IgG Fc fragment improves the in vivo duration of action of the drug and minimizes the in vivo activity reduction of the drug.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.14/327,064 filed Jul. 9, 2014 (issued as U.S. Pat. No. 10,272,159),which is a divisional of U.S. application Ser. No. 10/535,341 filed Jun.9, 2006 (issued as U.S. Pat. No. 8,846,874), which is a National Stageof International Application No. PCT/KR2004/002942, filed on Nov. 13,2004, which claims the benefit of priority from Korean PatentApplication No. 10-2003-0080299, filed on Nov. 13, 2003, the contents ofwhich are herein incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to an IgG Fc fragment useful as a drugcarrier, and more particularly, to IgG2 Fc and IgG4 Fc fragments,combinations thereof and hybrids thereof. Also, the present invention isconcerned with an expression vector for expressing the IgG Fc fragment,a transformant transformed with the said expression vector and a methodfor preparing an immunoglobulin Fc fragment by culturing the saidtransformant.

BACKGROUND ART

In the past, a large number of pharmacologists and chemists made effortsto chemically alter and/or modify the in vivo activity of naturallyexisting, physiologically active molecules. These efforts mainly focusedon increasing or prolonging certain in vivo activity, reducing toxicity,eliminating or reducing side effects, or modifying specificphysiological activities of the physiologically active substances. Whena physiologically active substance is chemically modified, it loses someor most of its physiological activities in many cases.

However, in some cases, the modification could result in an increase orchange in physiological activity. In this regard, many studies have beenfocused on chemical modification capable of achieving desiredphysiological activity, and most of such studies have involvedcovalently bonding a physiologically active substance (drug) to aphysiologically acceptable carrier.

For example, International Pat. Publication No. WO 01/93911 employs apolymer having a plurality of acid moieties as a drug carrier.International Pat. Publication No. WO 03/00778 discloses an anionicgroup-containing amphiphilic block copolymers that, when used as a drugcarrier for a cationic drug, improve the stability of the drug. EuropeanPat. No. 0 681 481 describes a method of improving the properties ofbasic drugs by using cyclodextrin and acids as carriers. On the otherhand, hydrophobic drugs have low stability in vivo mainly due their lowaqueous solubility. To improve the low aqueous solubility of hydrophobicdrugs, International Pat. Publication No. WO 04/064731 employs a lipidas a carrier. However, to date, there is no report for the use of aimmunoglobulin Fc fragment as a drug carrier.

Typically, since polypeptides are relatively easily denatured due totheir low stability, degraded by proteolytic enzymes in the blood andeasily eliminated through the kidney or liver, protein medicaments,including polypeptides as pharmaceutically effective components, need tobe frequently administered to patients to maintain desired blood levelconcentrations and titers. However, this frequent administration ofprotein medicaments, especially through injection causes pain forpatients. To solve these problems, many efforts have been made toimprove the serum stability of protein drugs and maintain the drugs inthe blood at high levels for a prolonged period of time, and thusmaximize the pharmaceutical efficacy of the drugs. Pharmaceuticalcompositions with sustained activity, therefore, need to increase thestability of protein drugs and maintain the titers at sufficiently highlevels without causing immune responses in patients.

To stabilize proteins and prevent enzymatic degradation and clearance bythe kidneys, a polymer having high solubility, such as polyethyleneglycol (hereinafter, referred to simply as “PEG”), was conventionallyused to chemically modify the surface of a protein drug. By binding tospecific or various regions of a target protein, PEG stabilizes theprotein and prevents hydrolysis, without causing serious side effects(Sada et al., J. Fermentation Bioengineering 71: 137-139, 1991).However, despite its capability to enhance protein stability, this PEGcoupling has problems such as greatly reducing the number titers ofphysiologically “active” proteins. Further the yield decreases with theincreasing molecular weight of PEG due to the reduced reactivity withthe proteins.

Recently, polymer-protein drug conjugates have been suggested. Forexample, as described in U.S. Pat. No. 5,738,846, a conjugate can beprepared by linking an identical protein drug to both ends of PEG toimprove the activity of the protein drug. Also, as described inInternational Pat. Publication No. WO 92/16221, two different proteindrugs can be linked to both ends of PEG to provide a conjugate havingtwo different activities. The above methods, however, were not verysuccessful in sustaining the activity of protein drugs.

On the other hand, Kinstler et al. reported that a fusion proteinprepared by coupling granulocyte-colony stimulating factor (G-CSF) tohuman albumin showed improved stability (Kinstler et al., PharmaceuticalResearch 12(12): 1883-1888, 1995). In this publication, however, sincethe modified drug, having a G-CSF-PEG-albumin structure, only showed anapproximately four-fold increase in residence time in the body and aslight increase in serum half-life compared to the single administrationof the native G-CSF, it has not been industrialized as effectivelong-acting formulation for protein drugs.

An alternative method for improving the in vivo stability ofphysiologically active proteins is by linking a gene of physiologicallyactive protein to a gene encoding a protein having serum stability bygenetic recombination technology and culturing the cells transfectedwith the recombinant gene to produce a fusion protein. For example, afusion protein can be prepared by conjugating albumin, a protein knownto be the most effective in enhancing protein stability, or its fragmentto a physiologically active protein of interest by genetic recombination(International Pat. Publication Nos. WO 93/15199 and WO 93/15200,European Pat. Publication No. 413,622). A fusion protein ofinterferon-alpha and albumin, developed by the Human Genome ScienceCompany and marketed under the trade name of ‘Albuferon™’, increased thehalf-life from 5 hours to 93 hours in monkeys, but it was known to beproblematic because it decreased the in vivo activity to less than 5% ofunmodified interferon-alpha (Osborn et al., J. Phar. Exp. Ther. 303(2):540-548, 2002). There has been no report of good technology that enhanceboth the in vivo duration of action and the stability of protein drugsas well as maintaining the in vivo physiological activity of the drugs.

On the other hand, immunoglobulins and their fragments were employed toenhance the stability of protein drugs. For example, U.S. Pat. No.5,045,312 discloses a method of increasing the activity of a growthhormone compared to an unmodified growth hormone by conjugating humangrowth hormone to serum albumin or rat immunoglobulin using acrosslinking agent. Also, other attempts were made to fuse a proteindrug to an immunoglobulin Fc fragment. For example, interferon (KoreanPat. Laid-open Publication No. 2003-9464), and interleukin-4 receptor,interleukin-7 receptor or erythropoietin (EPO) receptor (Korean Pat.Registration No. 249572) were previously expressed in mammals in a formfused to an immunoglobulin Fc fragment. International Pat. PublicationNo. WO 01/03737 describes a fusion protein comprising a cytokine orgrowth factor linked to an immunoglobulin Fc fragment through peptidelinkage. In addition, U.S. Pat. No. 5,116,964 discloses proteins fusedto the amino- or carboxyl-terminal end of an immunoglobulin Fc fragmentby genetic recombination. U.S. Pat. No. 5,349,053 discloses a fusionprotein comprising IL-2 fused to an immunoglobulin Fc fragment throughpeptide linkage.

Other examples of Fc fusion proteins prepared by genetic recombinationinclude a fusion protein of interferon-beta or its derivative and animmunoglobulin Fc fragment (International Pat. Publication NO. WO00/23472), and a fusion protein of IL-5 receptor and an immunoglobulinFc fragment (U.S. Pat. No. 5,712,121). However, techniques for improvingthe duration of action for physiologically active polypeptide drugsusing an immunoglobulin Fc fragment are mostly focused on using theimmunoglobulin Fc fragment only as a fusion partner, and to date, thetechnique of using an immunoglobulin Fc fragment as a carrier has notbeen reported.

Techniques involving the modification of amino acid residues of animmunoglobulin Fc fragment are also known. For example, U.S. Pat. No.5,605,690 discloses a TNFR-IgG1 Fc fusion protein, which is prepared bygenetic recombination using an IgG1 Fc fragment having amino acidalterations in the complement binding region or receptor binding region.

However, such Fc fusion proteins produced by genetic recombination havethe following disadvantages: protein fusion occurs only in a specificregion of an immunoglobulin Fc fragment, which is at an amino- orcarboxyl-terminal end; only homodimeric forms and not monomeric formsare produced; and a fusion could take place only between theglycosylated proteins or between the aglycosylated proteins, and it isimpossible to make a fusion protein composed of a glycosylated proteinand an aglycosylated protein. Further, a new amino acid sequence createdby the fusion may trigger immune responses, and a linker region maybecome susceptible to proteolytic degradation.

To solve these problems, the inventors of the present applicationconducted a research, and came to a knowledge that, when an IgG Fcfragment, more particularly an IgG2 or IgG4 Fc fragment, is linked to adrug, it could improve the in vivo duration of the drug and minimize areduction in the in vivo activity.

DISCLOSURE OF THE INVENTION

It is therefore an object of the present invention to provide animmunoglobulin G fragment that is useful as a drug carrier.

It is another object of the present invention to provide a recombinantvector expressing an immunoglobulin G fragment.

It is a further object of the present invention to provide atransformant transformed with a recombinant vector expressing animmunoglobulin G fragment.

It is yet another object of the present invention to provide a methodfor preparing an immunoglobulin G fragment, comprising culturing atransformant transformed with a recombinant vector expressing theimmunoglobulin G fragment.

It is still another object of the present invention to provide apharmaceutical composition comprising an immunoglobulin G fragment.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 shows the results of Western blotting of immunoglobulin Fcfragments expressed in E. coli under non-reduced conditions;

FIGS. 2 and 3 show the results of SDS-PAGE of immunoglobulin Fcfragments under non-reduced and reduced conditions using a 15% criteriongel (Bio-Rad);

FIG. 4 shows the results of chromatography of an immunoglobulin Fcfragment obtained by cleavage of an immunoglobulin with papain;

FIG. 5 shows the results of SDS-PAGE of a purified immunoglobulin Fcfragment (M: molecular size marker, lane 1: IgG, lane 2: Fc);

FIG. 6 shows the results of SDS-PAGE of IFNα-PEG-Fc (A),¹⁷Ser-G-CSF-PEG-Fc (B) and EPO-PEG-Fc (C) conjugates, which aregenerated by a coupling reaction (M: molecular size marker, lane 1: Fc,lane 2: physiologically active protein, lane 3: physiologically activeprotein-PEG-Fc conjugate);

FIG. 7 shows the results of size exclusion chromatography of anIFNα-PEG-Fc conjugate that is purified after a coupling reaction;

FIG. 8 shows the results of MALDI-TOF mass spectrometry of an EPO-PEG-Fcconjugate;

FIGS. 9a and 9b show the results of MALDI-TOF mass spectrometry andSDS-PAGE analysis, respectively, of a native immunoglobulin Fc and adeglycosylated immunoglobulin Fc (DG Fc);

FIG. 10 shows the results of MALDI-TOF mass spectrometry of anIFNα-PEG-Fc conjugate and an IFNα-PEG-DG Fc conjugate;

FIGS. 11a to 11c show the results of reverse phase HPLC of IFNα-PEG-Fc,IFNα-PEG-DG Fc and IFNα-PEG-recombinant AG Fc derivative conjugates;

FIG. 12 is a graph showing the results of pharmacokinetic analysis of anative IFNα, an IFNα-40K PEG complex, an IFNα-PEG-albumin conjugate andan IFNα-PEG-Fc conjugate;

FIG. 13 is a graph showing the results of pharmacokinetic analysis of anative EPO, a highly glycosylated EPO, an EPO-PEG-Fc conjugate and anEPO-PEG-AG Fc conjugate;

FIG. 14 is a graph showing the results of pharmacokinetic analysis ofIFNα-PEG-Fc, IFNα-PEG-DG Fc and IFNα-PEG-recombinant AG Fc conjugates;

FIG. 15 is a graph showing the pharmacokinetics of a Fab′, a Fab′-S-40KPEG complex, a Fab′-N-PEG-N-Fc conjugate and a Fab′-S-PEG-N-Fcconjugate;

FIG. 16 is a graph showing the in vivo activities of Fab′, a Fab′-S-40KPEG complex, a Fab′-N-PEG-N-Fc conjugate and a Fab′-S-PEG-N-Fcconjugate;

FIG. 17 is a graph showing the results of comparison of human IgGsubclasses for binding affinity to the C1q complement; and

FIG. 18 is a graph showing the results of comparison of a glycosylatedFc, an enzymatically deglycosylated DG Fc and an interferon-PEG-carrierconjugate where the carrier is AG Fc produced by E. coli for bindingaffinity to the C1q complement.

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention relates to an immunoglobulin G Fcfragment useful as a drug carrier, and more preferably IgG2 Fc and IgG4Fc fragments.

The term “carrier”, as used herein, refers to a substance linked with adrug, which typically increases, decreases or eliminates thephysiological activity of the drug by binding to the drug. However, withrespect to the objects of the present invention, a carrier is employedin the present invention for minimizing a decrease in the physiologicalactivity of a drug of interest, linked to the carrier, while enhancingthe in vivo stability of the drug.

A large number of substances, such as lipids and polymers, were studiedto determine their suitability as drug carriers. However, techniquesemploying an immunoglobulin Fc fragment as a drug carrier are unknown.That is, the present invention is characterized by providingparticularly an IgG Fc fragment among various substances available ascarriers for improving the in vivo duration of action of a drug to whichthe carrier is conjugated and minimizing decrease in the in vivoactivity of the drug and more preferably IgG2 Fc and IgG4 Fc fragments.

The term “immunoglobulin G (hereinafter, used interchangeably with“IgG”)”, a used herein, collectively means proteins that participate inthe body's protective immunity by selectively acting against antigens,and may be derived from humans and animals. Immunoglobulins have thefollowing general structure. Immunoglobulins are composed of twoidentical light chains and two identical heavy chains. The light andheavy chains comprise variable and constant regions. There are fivedistinct types of heavy chains based on differences in the amino acidsequences of their constant regions: gamma (γ), mu (μ), alpha (α), delta(δ) and epsilon (ε), and the heavy chains include the followingsubclasses: gamma 1 (γ1), gamma 2 (γ2) , gamma 3 (γ3), gamma 4 (γ4),alpha 1 (α1) and alpha 2 (α2). Also, there are two types of light chainsbased on differences in the amino acid sequences of their constantregions: kappa (κ) and lambda (λ) types (Coleman et al., FundamentalImmunology, 2nd Ed., 1989, 55-73). According to the features of theconstant regions of the heavy chains, immunoglobulins are classifiedinto five isotypes: IgG, IgA, IgD, IgE and IgM. IgG is divided intoIgG1, IgG2, IgG3 and G4 subclasses.

In addition, immunoglobulins are known to generate several structurallydifferent fragments, which include Fab, F (ab′), F (ab′) 2, Fv, scFv, Fdand Fc. Among the immunoglobulin fragments, Fab contains the variableregions of the light chain and the heavy chain, the constant region ofthe light chain and the first constant region (C_(H)1) of the heavychain, and has a single antigen-binding site. The Fab′ fragments differfrom the Fab fragments in terms of having the hinge region containingone more cysteine residues the C-terminus (carboxyl terminus) of theheavy chain C_(H)1 domain. The F (ab′)₂ fragments are produced as a pairof the Fab′ fragments by disulfide bonding formed between cysteineresidues of the hinge regions of the Fab′ fragments. Fv is the minimumantibody fragment that contains only heavy-chain variable region and thelight-chain variable region. The scFv (single-chain Fv) fragmentscomprise both the heavy-chain variable region and the light-chainvariable region that are linked to each other a peptide linker and thusare present in a single polypeptide chain. The Fd fragments compriseonly the variable region and C_(H)1 domain of the heavy chain.

Among the known various types of immunoglobulins and their functionaland structural fragments, as described above, the present invention ischaracterized by providing an IgG Fc fragment useful as a drug carrier,and more preferably IgG2 Fc and IgG4 Fc fragments.

The term “immunoglobulin G Fc fragment (hereinafter, usedinterchangeably with “IgG Fc fragment” or “Fc fragment of the presentinvention”)”, as used herein, refers to a protein that contains theheavy-chain constant region 2 (C_(H)2) and the heavy-chain constant 3(C_(H)3) of an immunoglobulin G, and not the variable regions of theheavy and light chains, the heavy-chain constant region 1 (C_(H)1) andthe light-chain constant region 1 (C_(L)1) of the immunoglobulin G. Itmay further include the hinge region at the heavy-chain constant region.Also, the IgG Fc fragment of the present invention may contain a portionor the all the heavy chain constant region 1 (C_(H)1) and/or thelight-chain constant region 1 (C_(L)1), except for the variable regionsof the heavy and light chains. Also, as long as it has a physiologicalfunction substantially similar to or better than the native protein theIgG Fc fragment may be a fragment having a deletion in a relatively longportion of the amino acid sequence of C_(H)2 and/or C_(H)3.

The Fc fragment of the present invention includes a native amino acidsequence and sequence derivatives (mutants) thereof. An amino acidsequence derivative is a sequence that is different from the nativeamino acid sequence due to a deletion, an insertion, a non-conservativeor conservative substitution or combinations thereof of one or moreamino acid residues. For example, in an IgG Fc, amino acid residuesknown to be important in binding, at positions 214 to 238, 297 to 299,318 to 322, or 327 to 331, may be used as a suitable target formodification. Also, other various derivatives are possible, includingone in which a region capable of forming a disulfide bond is deleted, orcertain amino acid residues are eliminated at the N-terminal end of anative Fc form or a methionine residue is added thereto. Further, toremove effector functions, a deletion may occur in a complement-bindingsite, such as a C1q-binding site and an ADCC site. Techniques ofpreparing such sequence derivatives of the immunoglobulin Fc fragmentare disclosed in International Pat. Publication Nos. WO 97/34631 and WO96/32478.

Amino acid exchanges in proteins and peptides, which do not generallyalter the activity of the proteins, or peptides are known the art (H.Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). Themost commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu,Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro,Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, in bothdirections.

In addition, the Fc fragment, if desired, may be modified byphosphorylation, sulfation, acrylation, glycosylation, methylation,farnesylation, acetylation, amidation, and the like.

The aforementioned Fc derivatives are derivatives that have biologicalactivity identical to the Fc fragment of the present invention orimproved structural stability, for example, against heat, pH, or thelike.

In addition, these Fc fragments may be obtained from native formsisolated from humans and other animals including cows, goats, swine,mice, rabbits, hamsters, rats and guinea pigs, or may be recombinants orderivatives thereof, obtained from transformed animal cells ormicroorganisms. Herein they may be obtained from a native immunoglobulinby isolating whole immunoglobulins from human or animal organisms andtreating them with a proteolytic enzyme. Papain digests the nativeimmunoglobulin into Fab and Fc fragments, and pepsin treatment resultsin the production of pF′c and F (ab′) 2 fragments. These fragments maybe subjected, for example, to size exclusion chromatography to isolateFc or pF′c. Preferably, a human-derived Fc fragment is a recombinant IgGFc fragment that is obtained from a microorganism. That is, preferredare human-derived recombinant IgG2 Fc and IgG4 Fc fragments obtainedfrom a microorganism.

In addition, the Fc fragment of the present invention may be in the formof having native sugar chains, increased sugar chains compared to anative form decreased sugar chains compared to the native form, or maybe in a deglycosylated form. The increase, decreases or removal of sugarchains of the Fc fragment may be achieved by methods common in the art,such as a chemical method, an enzymatic method and a genetic engineeringmethod using a microorganism. The removal of sugar chains from an Fcfragment results in a sharp decrease in binding affinity to the C1q partof the first complement component C1 and a decrease or loss inantibody-dependent cell-mediated cytotoxicity (ADCC) orcomplement-dependent cytotoxicity (CDC), thereby not inducingunnecessary immune responses in vivo. In this regard, an immunoglobulinFc fragment in a deglycosylated or aglycosylated form may be moresuitable to the object of the present invention as a drug carrier.

As apparent in FIG. 18, a glycosylated Fc has stronger CDC activity thanan aglycosylated Fc and thus has a high risk of inducing immuneresponses. Thus, with the objects of the present invention, preferred isan aglycosylated or deglycosylated Fc fragment. More preferred areaglycosylated IgG2 Fc and IgG4 Fc fragments, combinations thereof andhybrids thereof.

As used herein, the term “deglycosylation” refers to that sugar moietiesare enzymatically removed from an Fc fragment, and the term“aglycosylatlon” means that an Fc fragment is produced in anunglycosylated form by a prokaryote, preferably E. coli.

On the other hand, the term “combination”, as used herein, means thatpolypeptides encoding single-chain immunoglobulin Fc fragments of thesame origin are linked to a single-chain polypeptide of a differentorigin to form a dimer or multimer. That is, a dimer or multimer may beformed from two or more fragments selected from the group consisting ofIgG1 Fc, IgG2 Fc, IgG3 Fc and IgG4 Fc fragments.

The term “hybrid”, as used herein, means that sequences encoding two ormore immunoglobulin Fc fragments of different origin are present in asingle-chain immunoglobulin Fc fragment. In the present invention,various types of hybrids are possible. That is, domain hybrids may becomposed of one to four domains selected from the group consisting ofCH1, CH2, CH3 and CH4 of IgG1 Fc, IgG2 Fc, IgG3 Fc and IgG4 Fc, and mayinclude the hinge region.

On the other hand, as shown in the accompanying drawings of the presentinvention, FIGS. 17 and 18, among several subclasses of IgG, IgG4 hasthe lowest binding affinity to complement C1q. The decrease in bindingaffinity to the complement results in a decrease in or removal ofantibody-dependent cell-mediated cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC), and thus, unnecessary immuneresponses are not induced in vivo. IgG2 and IgG4 Fc fragments haveweaker binding affinity to C1q than IgG1, and the IgG4 Fc fragment hasthe weakest activity. Therefore, since, to be used as a drug carrier,the Fc fragment linked to a drug preferably has weaker effector functionactivities such as ADCC and CDC, with respect to the objects of thepresent invention, preferred are IgG2 Fc and IgG4 Fc fragments, morepreferred is the IgG4 Fc fragment, and most preferred are Fc fragmentshaving the amino acid sequences of SEQ ID Nos. 8, 10 and 23.

In another aspect, the present invention relates to a gene encoding anIgG Fc fragment, preferably genes encoding IgG2 Fc and IgG4 Fcfragments, and more preferably genes encoding the amino acid sequencesof SEQ ID Nos. 8, 10 and 23. Such a gene encoding the Fc of the presentinvention includes a native nucleotide sequence and sequence derivativesthereof. A nucleotide sequence derivative means to have a sequencedifferent by a deletion, an insertion, a non-conservative orconservative substitution in one or more nucleotide residues of a nativenucleotide sequence, or combinations thereof.

In the present invention, a gene encoding an Fc fragment having theamino acid sequence of SEQ ID No. 8 is preferably a gene having thenucleotide sequence of SEQ ID No. 4. A gene encoding an Fc fragmenthaving the amino acid sequence of SEQ ID No. 10 is preferably a genehaving the nucleotide sequence of SEQ ID No. 9. A gene encoding an Fcfragment having the amino acid sequence of SEQ ID No. 23 is preferably agene having the nucleotide sequence of SEQ ID No. 22. The nucleotidesequences encoding the Fc fragments of the present invention may bealtered by a substitution, a deletion or an insertion of one more bases,or combinations thereof. The nucleotide sequences may be naturallyisolated or artificially synthesized, or may be prepared by a geneticrecombination method.

The nucleotide sequences encoding the Fc fragments of the presentinvention are provided by vectors expressing the same.

In a further aspect, the present invention provides a recombinant vectorcomprising an IgG Fc fragment.

The term “vector”, as used herein, means a vehicle for introducing a DNAmolecule into a host cell to express an antibody or an antibodyfragment. The vector useful in the present invention includes plasmidvectors, cosmid vectors, bacteriophage vectors, and viral vectors suchas adenovirus vectors, retrovirus vectors and adeno-associated virusvectors. The plasmid vector is preferable. With respect to the objectsof the present invention, an expression vector may include expressionregulatory elements, such as a promoter, an initiation codon, a stopcodon, a polyadenylation signal and an enhancer, and a signal sequencefor membrane targeting or secretion.

The term “signal sequence”, as used herein, refers to a specific aminoacid sequence that allows transport and secretion of a protein to theoutside of the cytosol. Various types of these signal sequences areknown in the art, but, since the present invention preferably uses E.coli as a host cell, the signal sequence of the present invention ispreferably an E. coli-derived signal sequence, which an E. colisecretory protein possesses. Examples of E. coli-derived signalsequences include alkaline phosphatase, penicillinase, Ipp, heat-stableenterotoxin II, LamB, PhoE, PelB, OmpA and maltose binding protein. Mostpreferred is heat-stable enterotoxin II.

On the other hand, the initiation and stop codons are generallyconsidered to be a portion of a nucleotide sequence coding for animmunogenic target protein, are necessary to be functional in anindividual to whom a genetic construct has been administered, and mustbe in frame with the coding sequence. Promoters may be generallyconstitutive or inducible. Non-limiting examples of promoters availablein prokaryotic cells include lac, tac, T3 and T7 promoters. Non-limitingexamples of promoters available in eukaryotic cells include simian virus40 (SV40) promoter, mouse mammary tumor virus (MMTV) promoter, humanimmunodeficiency virus (HIV) promoter such as the HIV Long TerminalRepeat (LTR) promoter, moloney virus promoter, cytomegalovirus (CMV)promoter, Epstein Barr virus (EBV) promoter, rous sarcoma virus (RSV)promoter, as well as promoters from human genes such as human β-actin,human hemoglobin, human muscle creatine and human metalothionein. Inaddition, expression vectors include a selectable marker that allowsselection of host cells containing the vector, and replicable expressionvectors include a replication origin. Genes coding for products thatconfer resistance to antibiotics or drugs are used as general selectablemarkers. β-latamase gene (ampicillin resistance) and Tet gene(tetracycline resistance) may be used in prokaryotic cells, and neomycin(G418 or Geneticin), gpt (mycophenolic acid), ampicillin and hygromycinresistant genes may be used in eukaryotic cells. Dihydropholatereductase marker gene may be selected by methotrexate in a variety ofhosts. Genes coding for gene products of auxotrophic markers of hosts,for example, LEU2, URA3 and HIS3, are often used as selectable markersin yeasts. Also, available are viruses (e.g., vaculovirus) or phagevectors, and vectors that are able to integrate into the genome of hostcells, such as retrovirus vectors.

To prepare an IgG Fc fragment that coincide with the objects of thepresent invention, a vector is used, which carries a gene coding for theamino acid sequence of SEQ ID No. 8, 10 or 23. In the present invention,using pT14S1SH-4T20V22Q (Korean Pat. No. 38061) as a starting vector,the following two vectors are constructed: pSTIIdCG2Fc that carries agene designated as SEQ ID No. 22 encoding the amino acid sequence of SEQID No. 23, and pSTIIdCG4Fc that carries a gene designated as SEQ ID No.4 encoding the amino acid sequence of SEQ ID No. 8. Also, by performingPCR using the pSTIIdCG4Fc plasmid, a gene designated as SEQ ID No. 9encoding the amino acid sequence of SEQ ID No. 10 is obtained, the genehaving a deletion in the hinge region required for dimer formation froma gene amplified by the PCR, and a pSTIIG4Mo vector carrying the gene isthen constructed.

In yet another aspect, the present invention relates to a transformanttransformed with the recombinant vector.

Since expression levels and modification of proteins vary depending onhost cells, the most suitable host cell may be selected according to theintended use. Available host cells include, but are not limited to,prokaryotic cells such as Escherichia coli, Bacillus subtilis,Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. Inaddition, useful as host cells are lower eukaryotic cells, such as fungi(e.g., Aspergillus) and yeasts (e.g., Pichia pastoris, Saccharomycescerevisiae, Schizosaccharomyces, Neurospora crassa), insect cells, plantcells, and cells derived from higher eukaryotes including mammals.However, since the immunoglobulin Fc fragment is advantageously in anaglycosylated form with respect to the objects of the present invention,the prokaryotic host cells are preferable, and in particular, E. coli ismost preferable.

In the present invention, “transformation” and/or “transfection” intohost cells includes any methods by which nucleic acids can be introducedinto organisms, cells, tissues or organs, and, as known in the art, maybe performed by selecting suitable standard techniques according to hostcells. These methods include, but are not limited to, electroporation,protoplast fusion, calcium phosphate (CaPO₄) precipitation, calciumchloride (CaCl₂) precipitation, and agitation with silicon carbidefiber, Agrobacterium-mediated transformation, and PEG-, dextransulfate-, lipofectamine- and desiccation/inhibition-mediatedtransformation. For example, calcium treatment using calcium chloride orelectroporation is generally used in prokaryotic cells (Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press)). Transfection using Agrobacterium tumefaciensis used for transformation of specific plant cells (Shaw et al., 1983,Gene, 23:315; International Pat. Publication No. WO 89/05859). Formammalian cells having no cell walls, calcium phosphate precipitationmay be used (Graham et al, 1978, Virology, 52:456-457). The generallymethods and features of transformation into mammalian host cells aredescribed in U.S. Pat. No. 4,399,216. Transformation into yeasts istypically carried out according to the methods described by Van Solingenet al., J. Bact., 1977, 130:946, and Hsiao et al., Proc. Natl. Acad.Sci. (USA), 1979, 76:3829.

The expression vectors according to the present invention transformedinto host cells, and the resulting transformants of the presentinvention are designated as HM10932 (pSTIIdCG4Fc-introducedtransformant), HM10933 (pSTIIG4Mo-introduced transformant) and HM10936(pSTIIdCG2Fc-introduced transformant).

In still another aspect, the present invention provides a method ofpreparing an immunoglobulin fragment, comprising culturing atransformant transformed with a vector capable of expressing an IgG Fcfragment, and preferably IgG2 Fc or Ig4 Fc fragment, or a combinationthereof or a hybrid thereof, under suitable conditions.

In the method of preparing the immunoglobulin fragment, the culturing oftransformant may be performed using suitable media under suitableculture conditions, which are known in art. This culturing process maybe easily adjusted according to the strains selected by those skilled inthe art.

The immunoglobulin fragment of present invention, obtained by culturingthe transformant, may be used in an unpurified form, or may be usedafter being purified with high purities using various general methods,for example, dialysis, salt precipitation and chromatography. Amongthem, chromatography is most commonly used. As no rule is applicable toany case in selecting the type and sequence of used columns,chromatography may be selected according to the properties and culturemethod of target proteins of antibodies, for example, from ion exchangechromatography, size exclusion chromatography, affinity chromatographyand protein-A affinity column chromatography. In preferred embodimentsof the present invention, the immunoglobulin fragment is purified usinga protein-A affinity column, a SP sepharose FF column, and the like.

When the Fc fragment thus obtained is in the free form, it may beconverted into a salt form by a per se known method or a modifiedmethod. In contrast, when it is obtained in a salt form, the salt may beconverted into the free form or another salt by a per se known method ora modified method. Also, the Fc fragment as produced by a transformantmay be treated before or after purification with an appropriateprotein-modifying enzyme for arbitrary modification or partialpolypeptide removal. Examples of the protein-modifying enzyme useful inthe present invention include trypsin, chymotrypsin, arginineendopeptidase, protein kinase and glycosidase.

The Fc fragment of the present invention, prepared as described above,acts as a drug carrier and forms a conjugate with a drug.

The term “drug conjugate” or “conjugate”, as used herein, means that oneor more drugs are linked with one or more immunoglobulin Fc fragments.

The term “drug”, as used herein, refers to a substance displayingtherapeutic activity when administered to humans or animals, andexamples of the drug include, but are not limited to, polypeptides,compounds, extracts and nucleic acids. Preferred is a polypeptide drug.

The terms “physiologically active polypeptide”, “physiologically activeprotein”, “active polypeptide” “polypeptide drug” and “protein drug”, asused herein, are interchangeable in their meanings, and are featured inthat they are in a physiologically active form exhibiting various invivo physiological functions.

The polypeptide drug has a disadvantage of being unable to sustainphysiological action for a long period of time due to its property ofbeing easily denatured or degraded by proteolytic enzymes present in thebody. However, when the polypeptide drug is conjugated to theimmunoglobulin Fc fragment of the present invention to form a conjugate,the drug has increased structural stability and degradation half-life.Also, the polypeptide conjugated to the Fc fragment has a much smallerdecrease in physiological activity than other known polypeptide drugformulations. Therefore, compared to the in vivo bioavailability ofconventional polypeptide drugs, the conjugate of the polypeptide and theFc fragment according to the present invention is characterized byhaving markedly improved in vivo bioavailability. This is also clearlydescribed through embodiments of the present invention. That is, whenlinked to the Fc fragment of the present invention, IFNα, G-CSF, hGH andother protein drugs displayed an about two- to six-fold increase in vivobioavailability compared to their conventional forms conjugated to PEGalone or both PEG and albumin (Tables 8, 9 and 10).

On the other hand, the linkage of a protein and the Fc fragment of thepresent invention is featured in that it is not a fusion by aconventional recombination method. A fusion form of the immunoglobulinFc fragment and an active polypeptide used as a drug by a recombinationmethod is obtained in such a way that the polypeptide is linked to theN-terminus or C-terminus of the Fc fragment, and is thus expressed andfolded as a single polypeptide from a nucleotide sequence encoding thefusion form.

This brings about a sharp decrease in the activity of the resultingfusion protein because the activity of a protein as a physiologicallyfunctional substance is determined by the conformation of the protein.Thus, when a polypeptide drug is fused with Fc by a recombinationmethod, there is no effect with regard to in vivo bioavailability evenwhen the fusion protein has increased structural stability. Also, sincesuch a fusion protein is often misfolded and thus expressed as inclusionbodies, the fusion method is uneconomical in protein production andisolation yield. Further, when the active form of a polypeptide is in aglycosylated form, the polypeptide should be expressed in eukaryoticcells. In this case, Fc is also glycosylated, and this glycosylation maycause unsuitable immune responses in vivo.

That is, only the present invention makes it possible to produce aconjugate of a glycosylated active polypeptide and an aglycosylatedimmunoglobulin Fc fragment, and overcomes all of the above problems,including improving protein production yield, because the two componentsof the complex are individually prepared and isolated by the bestsystems.

Non-limiting examples of protein drugs capable of being conjugated tothe immunoglobulin Fc fragment of the present invention include humangrowth hormone, growth hormone releasing hormone, growth hormonereleasing peptide, interferons and interferon receptors (e.g.,interferon-α, -β and -γ, water-soluble type I interferon receptor,etc.), granulocyte colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), glucagon-likepeptides (e.g., GLP-1, etc.), G-protein-coupled receptor, interleukins(e.g., IL-1 receptor, IL-4 receptor, etc.), enzymes (e.g.,glucocerebrosidase, iduronate-2-sulfatase, alpha-galactosidase-A,agalsidase alpha and beta, alpha-L-iduronidase, butyrylcholinesterase,chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase,platelet-activating factor acetylhydrolase, neutral endopeptidase,myeloperoxidase, etc.), interleukin and cytokine binding proteins (e.g.,IL-18bp, TNF-binding protein, etc.), macrophage activating factor,macrophage peptide, B cell factor, T cell factor, protein A, allergyinhibitor, cell necrosis glycoproteins, immunotoxin, lymphotoxin, tumornecrosis factor, tumor suppressors, metastasis growth factor, alpha-1antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E,erythropoietin, highly glycosylated erythropoietin, angiopoietins,hemoglobin, thrombin, thrombin receptor activating peptide,thrombomodulin, factor VII, factor VIIa, factor VIII, factor IX, factorXIII, plasminogen activating factor, fibrin-binding peptide, urokinase,streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor,collagenase inhibitor, superoxide dismutase, leptin, platelet-derivedgrowth factor, epithelial growth factor, epidermal growth factor,angiostatin, angiotensin, bone growth factor, bone stimulating protein,calcitonin, insulin, atriopeptin, cartilage inducing factor, elcatonin,connective tissue activating factor, tissue factor pathway inhibitor,follicle stimulating hormone, luteinizing hormone, luteinizing hormonereleasing hormone, nerve growth factors (e.g., nerve growth factor,ciliary neurotrophic factor, axogenesis factor-1, brain-natriureticpeptide, glial derived neurotrophic factor, netrin, neurophil inhibitorfactor, neurotrophic factor, neuturin, etc.), parathyroid hormone,relaxin, secretin, somatomedin, insulin-like growth factor,adrenocortical hormone, glucagon, cholecystokinin, pancreaticpolypeptide, gastrin releasing peptide, corticotropin releasing factor,thyroid stimulating hormone, autotaxin, lactoferrin, myostatin,receptors (e.g., TNFR(P75), TNFR(P55), IL-1 receptor, VEGF receptor, Bcell activating factor receptor, etc.), receptor antagonists (e.g.,IL1-Ra etc.), cell surface antigens (e.g., CD 2, 3, 4, 5, 7, 11a, 11b,18, 19, 20, 23, 25, 33, 38, 40, 45, 69, etc.), monoclonal antibodies,polyclonal antibodies, antibody fragments (e.g., scFv, Fab, Fab′,F(ab′)2 and Fd), and virus derived vaccine antigens. An antibodyfragment may be Fab, Fab′, F (ab′) 2, Fd or scFv, which is capable ofbinding to a specific antigen, and preferably Fab′.

In particular, preferred as physiologically active polypeptides arethose requiring frequent dosing upon administration to the body fortherapy or prevention of diseases, which include human growth hormone,interferons (interferon-α, -β, -γ, etc.), granulocyte colony stimulatingfactor, erythropoietin (EPO) and antibody fragments. In addition,certain derivatives are included in the scope of the physiologicallyactive polypeptides of the present invention as long as they havefunction, structure, activity or stability substantially identical to orimproved compared over native forms of the physiologically activepolypeptides. In the present invention, the most preferable polypeptidedrug is interferon-alpha.

In addition to the polypeptide drugs, other drugs are also available inthe present invention. Non-limiting examples of these drugs includeantibiotics selected from among derivatives and mixtures oftetracycline, minocycline, doxycycline, ofloxacin, levofloxacin,ciprofloxacin, clarithromycin, erythromycin, cefaclor, cefotaxime,imipenem, penicillin, gentamycin, streptomycin, vancomycin, and thelike; anticancer agents selected from among derivatives and mixtures ofmethotrexate, carboplatin, taxol, cisplatin, 5-fluorouracil,doxorubicin, etoposide, paclitaxel, camtotecin, cytosine arabinoside,and the like; anti-inflammatory agents selected from among derivativesand mixtures of indomethacin, ibuprofen, ketoprofen, piroxicam,probiprofen, diclofenac, and the like; antiviral agents selected fromamong derivatives and mixtures of acyclovir and robavin; andantibacterial agents selected from among derivatives and mixtures ofketoconazole, itraconazole, fluconazole, amphotericin B andgriseofulvin.

On the other hand, the Fc fragment of the present invention is able toform a conjugate linked to a drug through a linker.

This linker includes peptide and non-peptide linkers. Preferred is anon-peptide linker, and more preferred is a non-peptide polymer. Thepeptide linker means amino acids, and preferably 1 to 20 amino acids,which are linearly linked to each other by peptide bonding, and may bein a glycosylated form. This peptide linker is preferably a peptidehaving a repeating unit of Gly and Ser, which is immunologicallyinactive for T cells. Examples of the non-peptide polymer include poly(ethylene glycol), poly (propylene glycol), copolymers of ethyleneglycol and propylene glycol, polyoxyethylated polyols, polyvinylalcohol, polysaccharides, dextran, polyvinyl ether, biodegradablepolymers such as PLA (poly (lactic acid) and PLGA (poly (lactic-glycolicacid)), lipid polymers, chitins, and hyaluronic acid. Most preferred ispoly (ethylene glycol) (PEG).

The conjugate of the present invention, Fc fragment-drug or Fcfragment-linker-drug, is made at various molar ratios. That is, thenumber of the Fc fragment and/or linker linked to a single polypeptidedrug is not limited. However, preferably, in the drug conjugate of thepresent invention, the drug and the Fc fragment are conjugated to eachother at a molar ratio of 1:1 to 10:1, and preferably 1:1 to 2:1.

In addition, the linkage of the Fc fragment of the present invention, acertain linker and a certain drug include all covalent bonds except fora peptide bond formed when the Fc fragment and a polypeptide drug areexpressed as a fusion protein by genetic recombination, and all types ofnon-covalent bonds such as hydrogen bonds, ionic interactions, van derWaals forces and hydrophobic interactions. However, with respect to thephysiological activity of the drug, the linkage is preferably made bycovalent bonds.

In addition, the Fc fragment of the present invention, a certain linkerand a certain drug may be linked to each other at a certain site of thedrug. On the other hand, the Fc fragment of the present invention and apolypeptide drug may be linked to each other at an N-terminus orC-terminus, and preferably at a free group, and a covalent bond betweenthe Fc fragment and the drug is easily formed especially at an aminoterminal end, an amino group of a lysine residue, an amino group of ahistidine residue, or a free cysteine residue.

On the other hand, the linkage of the Fc fragment of the presentinvention, a certain linker and a certain drug may be made in a certaindirection. That is, the linker may be linked to the N-terminus, theC-terminus or a free group of the immunoglobulin Fc fragment, and mayalso be linked to the N-terminus, the C-terminus or a free group of theprotein drug. When the linker is a peptide linker, the linkage may takeplace at a certain linking site.

Also, the conjugate of the present invention may be prepared using anyof a number of coupling agents known in the art. Non-limiting examplesof the coupling agents include 1,1-bis (diazoacetyl)-2-phenylethane,glutaradehyde, N-hydroxysuccinimide esters such as esters with4-azidosalicylic acid, imidoesters including disuccinimidyl esters suchas 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane.

On the other hand, the conjugate of the novel Fc fragment of the presentinvention and a drug may offer a various number of pharmaceuticalcompositions.

The term “administration”, as used herein, means introduction of apredetermined amount of a substance into a patient by a certain suitablemethod. The conjugate of the present invention may be administered viaany of the common routes, as long as it is able to reach a desiredtissue. A variety of modes of administration are contemplated, includingintraperitoneally, intravenously, intramuscularly, subcutaneously,intradermally, orally, topically, intranasally, intrapulmonarily andintrarectally, but the present invention is not limited to theseexemplified modes of administration. However, since peptides aredigested upon oral administration, active ingredients of a compositionfor oral administration should be coated or formulated for protectionagainst degradation in the stomach. Preferably, the present compositionmay be administered in an injectable form. In addition, thepharmaceutical composition of the present invention may be administeredusing a certain apparatus capable of transporting the active ingredientsinto a target cell.

The pharmaceutical composition comprising the conjugate according to thepresent invention may include a pharmaceutically acceptable carrier. Fororal administration, the pharmaceutically acceptable carrier may includebinders, lubricants, disintegrators, excipients, solubilizers,dispersing agents, stabilizers, suspending agents, coloring agents andperfumes. For injectable preparations, the pharmaceutically acceptablecarrier may include buffering agents, preserving agents, analgesics,solubilizers, isotonic agents and stabilizers. For preparations fortopical administration, the pharmaceutically acceptable carrier mayinclude bases, excipients, lubricants and preserving agents. Thepharmaceutical composition of the present invention may be formulatedinto a variety of dosage forms in combination with the aforementionedpharmaceutically acceptable carriers. For example, for oraladministration, the pharmaceutical composition may be formulated intotablets, troches, capsules, elixirs, suspensions, syrups or wafers. Forinjectable preparations, the pharmaceutical composition may beformulated into a unit dosage form, such as a multidose container or anampule as a single-dose dosage form. The pharmaceutical composition maybe also formulated into solutions, suspensions, tablets, capsules andlong-acting preparations.

On the other hand, examples of carriers, exipients and diluents suitablefor the pharmaceutical formulations include lactose, dextrose, sucrose,sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acaciarubber, alginate, gelatin, calcium phosphate, calcium silicate,cellulose, methylcellulose, microcrystalline cellulose,polyvinylpyrrolidone, water, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate and mineral oils. Inaddition, the pharmaceutical formulations may further include fillers,anti-coagulating agents, lubricants, humectants, perfumes, emulsifiersand antiseptics.

A substantial dosage of a drug in combination with the Fc fragment ofthe present invention as a carrier may be determined by several relatedfactors including the types of diseases to be treated, administrationroutes, the patient's age, gender, weight and severity of the illness,as well as by the types of the drug as an active component. Since thepharmaceutical composition of the present invention has a very longduration of action in vivo, it has an advantage of greatly reducingadministration frequency of pharmaceutical drugs.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

EXAMPLES Preparation of Immunoglobulin Fc Fragments Example 1Construction of Human Immunoglobulin IgG4 Fc Expression Vector

<1-1> Construction of Dimeric IgG4 Fc Expression Vector

To clone a gene encoding the Fc region of human immunoglobulin IgG4,RT-PCR was carried out using RNA isolated from human blood cells as atemplate, as follows. First, total RNA was isolated from about 6 ml ofblood using a Qiamp RNA blood kit (Qiagen), and gene amplification wasperformed using the total RNA as a template and a One-Step RT-PCR kit(Qiagen). To obtain a desired gene sequence, a pair of primersrepresented by SEQ ID Nos. 1 and 2 was used. SEQ ID No. 1 is anucleotide sequence starting from the 10th residue, serine, of 12 aminoacid residues of the hinge region of IgG4 (Glu Ser Lys Tyr Gly Pro ProCys Pro Ser Cys Pro: SEQ ID No. 3). SEQ ID No. 2 was designed to have aBamHI recognition site containing a stop codon. The gene amplified usingthe primer set was identified to have the nucleotide sequencerepresented by SEQ ID No. 4 and contain an amino terminal end, startingwith the Ser-Cys-Pro sequence of the hinge region of a full-length IgG4Fc gene sequence, and CH2 and CH3 domains. To clone the amplified IgG4Fc gene fragment into an expression vector containing an E. coli signalsequence, an expression vector pT14S1SH-4T20V22Q (Korean Pat. No.38061), previously developed by the present inventors, was used as astarting vector. This expression vector contains an E. coli heat-stableenterotoxin signal sequence derivative having the nucleotide sequencerepresented by SEQ ID No. 5. To facilitate cloning, a StuI recognitionsite was inserted into an end of the E. coli heat-stable enterotoxinsignal sequence derivative of the pT14S1SH-4T20V22Q plasmid throughsite-directed mutagenesis using a pair of primers represented by SEQ IDNos. 6 and 7 to induce mutagenesis to introduce the StuI site at anucleotide sequence coding for the last amino acid residue of the signalsequence. This insertion of the StuI site was identified to besuccessful by DNA sequencing. The resulting pT14S1SH-4T20V22Q plasmidcontaining a StuI site was designated as “pmSTII”. The pmSTII plasmidwas treated with StuI and BamHI and subjected to agarose gelelectrophoresis, and a large fragment (4.7 kb), which contained the E.coli heat-stable enterotoxin signal sequence derivative, was purified.Then, the amplified IgG4 Fc gene fragment was digested with BamHI andligated with the linearized expression vector, thus providing apSTIIdCG4Fc plasmid. This vector expresses a protein that has the aminoacid sequence of SEC ID No. 8 and is present in a dimeric form bydisulfide bonds between cysteine residues in the hinge region. The finalexpression vector was transformed into E. coli BL21 (DE3), and theresulting transformant was designated as “BL21/pSTIIdCG4Fc (HM10932)”,which was deposited at the Korean Culture Center of Microorganisms(KCCM) on Sep. 15, 2004 and assigned accession number KCCM-10597.

<1-2> Construction of Monomeric IgG4 Fc Expression Vector

To clone an IgG4 Fc fragment to be expressed in a monomeric form, PCRwas carried out using a pair of primers represented by SEQ ID Nos. 9 and2 and the pSTIIdCG4Fc plasmid prepared in the above <1-1> as a template.To allow an amplified gene to be expressed in a monomeric form, the PCRwas designed for a PCR product to have a deletion in the hinge regionrequired for dimer formation from the IgG4 Fc sequence, and thus, onlythe CH2 and CH3 domains of IgG4 Fc were amplified. The PCR product wascloned into an expression vector, pmSTII, according to the sameprocedure as in the above <1-1>, thus providing a pSTIIG4Mo plasmid.This expression vector was transformed into E. coli BL21 (DE3), and theresulting transformant was designated as “BL21/pSTIIdCG4Mo (HM10933)”,which was deposited at the Korean Culture Center of Microorganisms(KCCM) on Sep. 15, 2004 and assigned accession number KCCM-10598. Aprotein expressed by the expression vector has the amino acid sequenceof SEQ ID No. 10, and is expressed from the CH2 domain and present in amonomeric form because it has no hinge region.

Example 2 Construction of Human Immunoglobulin IgG1 Fc Expression Vector

<2-1> Construction of Dimeric IgG1 Fc Expression Vector

To clone a gene encoding the Fc region of human IgG1, RT-PCR was carriedout according to the same method as in the <1-1> of Example 1 using RNAisolated from human blood cells as a template using a One-Step RT-PCRkit (Qiagen). To obtain a desired gene sequence, a pair of primersrepresented by SEQ ID Nos. 11 and 12 was used.

SEQ ID No. 11 is a nucleotide sequence starting from the 13th residue,proline, of 15 amino acid residues of the hinge region (Glu Pro Lys SerCys Asp Lys Thr His Thr Cys Pro Pro Cys Pro: SEQ ID No. 13).

The gene amplified using the pair of primers represented by SEQ ID Nos.11 and 12 was found to contain an amino terminal end starting with thePro-Cys-Pro sequence of the hinge region and CH2 and CH3 domains, amonga full-length IgG1 Fc gene sequence, and has the nucleotide sequence ofSEQ ID No. 14.

To clone the amplified IgG1 Fc gene into an expression vector containingan E. coli signal sequence, the aforementioned pmSTII vector was used.According to a similar cloning procedure to that in the <1-1> of Example1, the pmSTII plasmid was treated with StuI and BamHI and subjected toagarose gel electrophoresis, and a large fragment (4.7 kb), whichcontained the E. coli heat-stable enterotoxin signal sequencederivative, was purified. Then, the amplified IgG1 Fc gene was digestedwith BamHI and ligated with the linearized expression vector, thusproviding pSTIIG1Fc. This vector expresses in a host cell a protein thathas the amino acid sequence of SEQ ID No. 15 and is present in a dimericform by disulfide bonds between cysteine residues in the hinge region.The final expression vector was transformed into E. coli BL21 (DE3), andthe resulting transformant was designated as “BL21/pSTIIdCG1Fc(HM10927)”, which was deposited at the Korean Culture Center ofMicroorganisms (KCCM) on Sep. 15, 2004 and assigned accession numberKCCM-10588.

<2-2> Construction of Monomeric IgG1 Fc Expression Vector

To prepare an IgG1 Fc fragment to be expressed in a monomeric form, PCRwas carried out using a pair of primers represented by SEQ ID Nos. 16and 12 and the pSTIIG1Fc plasmid prepared in the <2-1> of Example 2 as atemplate. The PCR product was cloned into an expression vector, pmSTII,according to the same procedure as in the <2-1> of Example 2, thusproviding a pSTIIG1Mo plasmid containing the nucleotide sequencerepresented by SEQ ID No. 17. This expression vector was transformedinto E. coli BL21 (DE3), and the resulting transformant was designatedas “BL21/pSTIIdCG1Mo (HM10930)”, which was deposited at the KoreanCulture Center of Microorganisms (KCCM) on Sep. 15, 2004 and assignedaccession number KCCM-10595. A protein expressed by the expressionvector is expressed from the CH2 domain and presents in a monomeric formbecause it was deleted from the hinge region containing cysteineresidues allowing dimer formation, and has the amino acid sequence ofSEQ ID No. 18.

Example 3 Construction of Human Immunoglobulin IgG2 Fc Expression Vector

To clone a gene encoding the Fc region of human IgG2, RT-PCR was carriedout according to the same method as in the <1-1> of Example 1 using RNAisolated from human blood cells as a template using a One-Step RT-PCRkit (Qiagen). To obtain a desired gene sequence, a pair of primersrepresented by SEQ ID Nos. 19 and 20 was used. SEQ ID No. 19 is anucleotide sequence starting from the 10th residue, proline, of 12 aminoacid residues of the hinge region (Glu Arg Lys Cys Cys Val Glu Cys ProPro Cys Pro: SEQ ID No. 21). The gene amplified using the pair ofprimers represented by Nos. 19 and 20 was identified to contain an aminoterminal end, starting with the Pro-Cys-Pro sequence of the hinge regionof a full-length IgG2 Fc gene sequence, and CH2 and CH3 domains, and hasthe nucleotide sequence of SEQ ID No. 22. To clone the amplified IgG2 Fcgene fragment into an expression vector containing an E. coli signalsequence, the aforementioned pmSTII vector was used. According to asimilar cloning procedure to that in the <1-1> of example 1, the pmSTIIplasmid was treated with StuI and BamHI and subjected to agarose gelelectrophoresis, and a large fragment (4.7 kb), which contained the E.coli heat-stable enterotoxin signal sequence derivative, was purified.Then, the amplified IgG1 Fc gene fragment was digested with BamHI andligated with the linearized expression vector fragment, thus providingpSTIIG2Fc. This vector expresses in a host cell a protein that has theamino acid sequence of SEQ ID No. 23 and is present in a dimeric form bydisulfide bonds between cysteine endues in the hinge region. The finalexpression vector was transformed into E. coli BL21 and the resultingtransformant was designated as BL21/pSTIIdCG2Fc (HM10936).

Example 4 Expression and Purification of Immunoglobulin Fc

<4-1> Evaluation of Expression of Immunoglobulin Fc

Bacterial transformants prepared in Examples 1, 2 and 3 wereindividually inoculated in a fermenter (Marubishi Company) and allowedto ferment, and were evaluated to determine whether they expressimmunoglobulin Fc fragments.

First, each transformant was grown in 100 ml of LB medium with agitationovernight and inoculated in the fermentor for large-scale culture. Thefermentor was maintained at 30° C. or 35° C. To prevent conversion froman aerobic to anaerobic environment, the cultures were aerated with20-vvm air and stirred at 500 rpm. To compensate for the insufficientnutrients for bacterial growth during fermentation, the cultures weresupplemented with glucose and yeast extracts according to the fermentedstates of bacteria. When the cultures reached an OD₆₀₀ value of 80-100,an inducer, IPTG, was added to the cultures in an amount of 20 μM to 4mM to induce protein expression. The cultures were further cultured for40 to 45 hrs until the OD value at 600 nm increased to 100 to 120.

The expression of immunoglobulin Fc in the E. coli transformants and theexpressed sites, water solubility and dimer formation of the expressedIg Fc were examined as follows. To determine whether an expressedproduct is secreted to the fermentation fluid or the periplasmic spaceof E. coli by the signal sequence fused to the expression vector, thefermentation fluid was centrifuged to obtain a cell-free fermentationfluid and collect cells. The cell-free fermentation fluid and aperiplasmic space solution obtained by osmotic shock of the collectedcells were subjected to Western blot analysis. As a result, a very smallamount of immunoglobulin Fc was detected. To investigate intracellularexpression of Ig Fc, cells were disrupted using an ultrasonicator(Misonix Company). The resulting cell lysate was centrifuged to separatewater-soluble substances from water-insoluble substances, and thewater-soluble substances were subjected to Western blot analysis, asfollows. The water-soluble substances were mixed with a protein samplebuffer not containing a reducing agent such as DTT or β-mercaptoethanol,and separated on a 15% SDS-PAGE gel (Criterion Gel, Bio-Rad). Then,proteins were transferred onto a nitrocellulose membrane and detectedwith an HRP-conjugated anti-human Fc antibody (Sigma). As shown in FIG.1, immunoglobulin Fc was overexpressed in a water-soluble form andlocated in the cytosol of E. coli. Also, products, expressed bytransformants transformed with expression vectors expressing Ig Fchaving a portion of a hinge region, were expressed as dimers. In FIG. 1,lanes 1, 2 and 3 show products expressed in HM10927, HM10932 andHM10936, respectively, and lane 4 shows Fc generated by papain treatmentof immunoglobulins produced in animal cells, which showed a slightlylarger size due to its sugar moieties on the SDS-PAGE gel than thatproduced in E. coli.

<4-2> N-Terminal Sequence Analysis

The water-soluble dimeric Ig Fc fragments, which were located in thecytosol of E. coli as demonstrated in the above <4-1>, were designed tobe translated in a fused form to a signal sequence. Thus, to determinewhether the Ig Fc fragments are located in E. coli cytosol in a formfused to the signal sequence when not secreted without signal sequenceprocessing, N-terminal amino acid sequences of the Ig Fc fragments weredetermined by the Basic Science Research Institute, Seoul, Korea.Samples used in the N-terminal amino acid sequence analysis wereprepared as follows.

First, a PVDF membrane (Bio-Rad) was immersed in methanol for about 2-3sec to be activated, and was sufficiently wet with a blocking buffer(170 mM glycine, 25 mM Tris-HCl (pH 8.0), 20% methanol). The proteinsamples separated on a non-reduced SDS-PAGE gel, prepared in the above<4-1>, were blotted onto a PVDF membrane for about one hour using ablotting kit (Hoefer Semi-Dry Transfer unit, Amersham). Proteinstransferred onto the PVDF membrane were stained with a protein dye,Coomassie Blue R-250 (Amnesco), for 3-4 sec, and washed with adestaining solution (water:acetic acid:methanol=5:1:4). Then, regionscontaining proteins from the membrane were cut out with scissors andsubjected to N-terminal sequence analysis.

As a result, the IgG1 Fc protein was found to have an N-terminalsequence of Pro-Cys-Pro-Ala-Pro-Glu-Leu-Leu-Gly-Gly (SEQ ID NO: 24), theIgG4 Fc protein had an N-terminal sequence ofSer-Cys-Pro-Ala-Pro-Glu-Phe-Leu-Gly-Gly (SEQ ID NO: 25) and the IgG2 Fcprotein had an N-terminal sequence ofPro-Cys-Pro-Ala-Pro-Pro-Val-Ala-Gly-Pro (SE ID NO: 26). As apparent fromthese results, the Fc fragments expressed by the E. coli transformantsof the present invention were found to have an accurate N-terminalsequence. These results indicate that, when expressed in a form fused toa signal sequence, the Fc fragments are not secreted to theextracellular membrane or periplasmic space, are accurately processed inthe signal sequence even upon overexpression and are present in awater-soluble form in the cytosol.

<4-3> Purification of Immunoglobulin Fc

Immunoglobulin Fc was purified using a protein-A affinity column knownto have strong affinity to immunoglobulins, as follows.

E. coli cells collected by centrifuging fermentation fluids weredisrupted by a microfluizer (Microfludics) to give cell lysates. Thecell lysates were subjected to two-step column chromatography to purifyrecombinant immunoglobulin Fc fragments present in the cytosol. 5 ml ofa protein-A affinity column (Pharmacia) was equilibrated with PBS, andthe cell lysates were loaded onto the column at a flow rate of 5 ml/min.Unbound proteins were washed out with PPS, and bound proteins wereeluted with 100 mM citrate (pH 3.0). The collected fractions weredesalted using a HiPrep 26/10 desalting column (Pharmacia) with 10 mMTris buffer (pH 8.0). Then, secondary anion exchange columnchromatography was carried out using 50 ml a Q HP 26/10 column(Pharmacia). The primary purified recombinant immunoglobulin Fcfractions were loaded the Q-Sepharose 26/10 column, and the column waseluted with a linear gradient of 0-0.2 NaCl in 10 mM Tris buffer (pH8.0), thus providing highly pure fractions. After being partiallypurified using the protein-A affinity column, expression levels of therecombinant Ig Fc fragments were determined as follows.

Protein expression levels after Expression vectors Transformants proteinA purification (mg/l) pSTIIdCG1Fc HM10927 400 pSTIIG1Mo HM10930 500pSTIIdCG4Fc HM10932 400 pSTIIG4Mo HM10933 600 pSTIIdCG2Fc HM10936 100

Since the immunoglobulin Fc proteins thus obtained are present indimeric or monomeric form of the heavy chain, they have differentmigration patterns on reduced SDS-PAGE and non-reduced SDS-PAGE. Theresults of SDS-PAGE analysis, formed to determine protein purities afterexpressed products were purified, are given in FIGS. 2 and 3.

FIGS. 2 and 3 show the results of SDS-PAGE analysis of the purifiedimmunoglobulin Fc fragments in a dimeric or monomeric form undernon-reduced and reduced conditions using a criterion gel (Bio-Rad),wherein the Fc fragments were evaluated for differential migration onreduced versus non-reduced gels. In FIG. 2, the A region shows proteinsseparated on a non-reduced SDS-PAGE gel, and the B region shows proteinson a reduced SDS-PAGE gel. Lane M indicates a prestained low-rangestandard protein marker (Bio-Rad), and lanes 1 to 4 indicate proteinsamples for immunoglobulin Fc produced by E. coli transformants,HM10927, HM10928 (deposited at the Korean Culture Center ofMicroorganisms (KCCM) on Sep. 15, 2004 and assigned accession numberKCCM-10589), HM10929 (deposited at KCCM on Sep. 15, 2004 and assignedaccession number KCCM-10594) and HM10932, respectively. As shown in FIG.2, on reduced SDS-PAGE, the Ig Fc fragments were present in a monomericform because disulfide bonds formed between cysteine residues of thehinge region were reduced, and were thus migrated the monomer distance.In contrast, on non-reduced SDS-PAGE, the Ig Fc fragments were presentin a dimeric form by disulfide bonds and thus had a migration distanceof about 42 kDa.

In FIG. 3, the A region shows proteins separated on a non-reducedSDS-PAGE gel, and the B region shows proteins on a reduced SDS-PAGE gel.Lane M indicates the standard protein marker, and lanes 1 and 2 indicateprotein samples for immunoglobulin Fc produced by E. coli transformants,HM10930 and HM10933, respectively. As shown in FIG. 3, the proteins didnot show a large difference in migration on reduced versus non-reducedgels, and only displayed a slightly different migration due to thereduction of intramolecular disulfide bonds.

Preparation of Conjugate of Immunoglobulin Fc and Drug

Example 5 Preparation I of IFNα-PEG-Immunoglobulin Fc Fragment Conjugate

<Step 1> Preparation of Immunoglobulin Fc Fragment Using Immunoglobulin

An immunoglobulin Fc fragment was prepared as follows. 200 mg of 150-kDaimmunoglobulin G (IgG) (Green Cross, Korea) dissolved in 10 mM phosphatebuffer was treated with 2 mg of a proteolytic enzyme, papain (Sigma) at37° C. for 2 hrs with gentle agitation. After the enzyme reaction, theimmunoglobulin Fc fragment regenerated thus was subjected tochromatography for purification using sequentially a Superdex column, aprotein A column and a cation exchange column. In detail, the reactionsolution was loaded onto a Superdex 200 column (Pharmacia) equilibratedwith 10 mM sodium phosphate buffer (PBS, pH 7.3), and the column waseluted with the same buffer at a flow rate of 1 ml/min. Unreactedimmunoglobulin molecules (IgG) and F(ab′)2, which had a relatively highmolecular weight compared to the immunoglobulin Fc fragment, wereremoved using their property of being eluted earlier than the Ig Fcfragment. Fab fragments having a molecular weight similar to the Ig Fcfragment were eliminated by protein A column chromatography (FIG. 4).The resulting fractions containing the Ig Fc fragment eluted from theSuperdex 200 column were loaded at a flow rate of 5 ml/min onto aprotein A column (Pharmacia) equilibrated with 20 mM phosphate buffer(pH 7.0), and the column was washed with the same buffer to removeproteins unbound to the column. Then, the protein A column was elutedwith 100 mM sodium citrate buffer (pH 3.0) to obtain highly pureimmunoglobulin Fc fragment. The Fc fractions collected from the proteinA column were finally purified using a cation exchange column (polyCAT,PolyLC Company), wherein this column loaded with the Fc fractions waseluted with a linear gradient of 0.15-0.4 M NaCl in 10 mM acetate buffer(pH 4.5), thus providing highly pure Fc fractions. The highly pure Fcfractions were analyzed by 12% SDS-PAGE (lane 2 in FIG. 5).

<Step 2> Preparation of IFNα-PEG Complex

3.4-kDa polyethylene glycol having an aldehyde reactive group at bothends, ALD-PEG-ALD (Shearwater), was mixed with human interferon alpha-2b(hIFNα-2b, MW: 20 kDa) dissolved in 100 mM phosphate buffer in an amountof 5 mg/ml at an IFNα:PEG molar ratio of 1:1, 1:2.5, 1:5, 1:10 and 1:20.To this mixture, a reducing agent, sodium cyanoborohydride (NaCNBH₃,Sigma), was added at a final concentration of 20 mM and was allowed toreact at 4° C. for 3 hrs with gentle agitation to allow PEG to link tothe amino terminal end of interferon alpha. To obtain a 1:1 complex ofPEG and interferon alpha, the reaction mixture was subjected to sizeexclusion chromatography using a Superdex® column (Pharmacia). TheIFNα-PEG complex was eluted from the column using 10 mM potassiumphosphate buffer (pH 6.0) as an elution buffer, and interferon alpha notlinked to PEG, unreacted PEG and dimer byproducts where PEG was linkedto two interferon alpha molecules were removed. The purified IFNα-PEGcomplex was concentrated to 5 mg/ml. Through this experiment, theoptimal reaction molar ratio for IFNα to PEG, providing the highestreactivity and generating the smallest amount of byproducts such asdimers, was found to be 1:2.5 to 1:5.

<Step 3> Preparation of IFNα-PEG-Fc Conjugate

To link the IFNα-PEG complex purified in the above step 2 to theN-terminus of an immunoglobulin Fc fragment, the immunoglobulin Fcfragment (about 53 kDa) prepared in the above step 1 was dissolved in 10mM phosphate buffer and mixed with the IFNα-PEG complex at an IFNα-PEGcomplex:Fc molar ratio of 1:1, 1:2, 1:4 and 1:8. After the phosphatebuffer concentration of the reaction solution was adjusted to 100 mM, areducing agent, NaCNBH₃, was added to the reaction solution at a finalconcentration of 20 mM and was allowed to react at 4° C. for 20 hrs withgentle agitation. Through this experiment, the optimal reaction molarratio for IFNα-PEG complex to Fc, providing the highest reactivity andgenerating the fewest byproducts such as dimers, was found to be 1:2.

<Step 4> Isolation and Purification of the IFNα-PEG-Fc Conjugate

After the reaction of the above step 3, the reaction mixture wassubjected to Superdex size exclusion chromatography so as to eliminateunreacted substances and byproducts and purify the IFNα-PEG-Fc proteinconjugate produced. After the reaction mixture was concentrated andloaded onto a Superdex column, 10 mM phosphate buffer (pH 7.3) waspassed through the column at a flow rate of 2.5 ml/min to remove unboundFc and unreacted substances, followed by column elution to collectIFNα-PEG-Fc protein conjugate fractions. Since the collected IFNα-PEG-Fcprotein conjugate fractions contained a small amount of impurities,unreacted Fc and interferon alpha dimers, cation exchange chromatographywas carried out to remove the impurities. The IFNα-PEG-Fc proteinconjugate fractions were loaded onto a PolyCAT LP column (PolyLC)equilibrated with 10 mM sodium acetate (pH 4.5), and the column waseluted with a linear gradient of 0-0.5 M NaCl in 10 mM sodium acetatebuffer (pH 4.5) using 1 M NaCl. Finally, the IFNα-PEG-Fc proteinconjugate was purified using an anion exchange column. The IFNα-PEG-Fcprotein conjugate fractions were loaded onto a PolyWAX LP column(PolyLC) equilibrated with 10 mM Tris-HCl (pH 7.5), and the column wasthen eluted with a linear gradient of 0-0.3 M NaCl in 10 mM Tris-HCl (pH7.5) using 1 M NaCl, thus isolating the IFNα-PEG-Fc protein conjugate ina highly pure form.

Example 6 Preparation II of IFNα-PEG-Fc Protein Conjugate

<Step 1> Preparation of Fc-PEG Complex

3.4-kDa polyethylene glycol having an aldehyde reactive group at bothends, ALD-PEG-ALD (Shearwater), was mixed with the immunoglobulin Fcfragment prepared in the step 1 of Example 5 at Fc:PEG molar ratios of1:1, 1:2.5, 1:5, 1:10 and 1:20, wherein the Ig Fc fragment had beendissolved in 100 mM phosphate buffer in an amount of 15 mg/ml. To thismixture, a reducing agent, NaCNBH₃ (Sigma), was added at a finalconcentration of 20 mM and was allowed to react at 4° C. for 3 hrs withgentle agitation. To obtain a 1:1 complex of PEG and Fc, the reactionmixture was subjected to size exclusion chromatography using a Superdex®column (Pharmacia). The Fc-PEG complex was eluted from the column using10 mM potassium phosphate buffer (pH 6.0) as an elution buffer, andimmunoglobulin Fc fragment not linked to PEG, unreacted PEG and dimer byproducts where PEG was linked to immunoglobulin Fc fragment moleculeswere removed. The purified Fc-PEG complex was concentrated to about 15mg/ml. Through this experiment, the optimal reaction molar ratio for Fcto PEG, providing the highest reactivity and generating the fewestbyproducts such as dimers, was found to be 1:3 to 1:10.

<Step 2> Formation and Purification of Conjugate of the Fc-PEG Complexand Interferon Alpha

To link the Fc-PEG complex purified in the above step 1 to theN-terminus of IFNα, the Fc-PEG complex was mixed with IFNα dissolved in10 mM phosphate buffer at Fc-PEG complex:IFNα molar ratios of 1:1,1:1.5, 1:3 and 1:6. After the phosphate buffer concentration of thereaction solution was adjusted to 10 mM, a reducing agent, NaCNBH₃, wasadded to the reaction solution at a final concentration of 20 mM and wasallowed to react at 4° C. for 20 hrs with gentle agitation. After thereaction was completed, unreacted substances and products were removedaccording to the same purification method in the step 4 of Example 5,thus isolating the Fc-PEG-IFNα protein conjugate in a highly pure form.

Example 7

Preparation of hGH-PEG-Fc Conjugate

An hGH-PEG-Fc conjugate was prepared and purified according to the samemethod as in Example 5, except that drug other than interferon alpha,human growth hormone (hGH, MW: 22 kDa) was used and a hGH:PEG molarratio was 1:5.

Example 8 Preparation of (G-CSF)-PEG-Fc Conjugate

A (G-CSF)-PEG-Fc conjugate was prepared and purified according to thesame method as in Example 5, except that drug other than interferonalpha, human granulocyte colony stimulating factor (G-CSF), was used andan G-CSF:PEG molar ratio was 1:5.

On the other hand, a ¹⁷S-G-CSF-PEG-Fc protein conjugate was prepared andpurified according to the same method as described above using a G-CSFderivative, ¹⁷S-G-CSF, having a serine substitution at the seventeenthamino acid residue of the native G-CSF.

Example 9 Preparation of EPO-PEG-Fc Conjugate

An EPO-PEG-Fc conjugate was prepared and purified according to the samemethod as in Example 5, except that drug other than interferon alpha,human erythropoietin (EPO), was used and an EPO:PEG molar ratio was 1:5.

Example 10 Preparation of Protein Conjugate Using PEG Having DifferentReactive Group

An IFNα-PEG-Fc protein conjugate was prepared using PEG having asuccinimidyl propionate (SPA) reactive group at both ends, as follows.3.4-kDa polyethylene glycol, SPA-PEG-SPA (Shearwater), was mixed with 10mg of interferon alpha dissolved in 100 mM phosphate buffer at IFNα:PEGmolar ratios of 1:1, 1:2.5, 1:5, 1:10 and 1:20. The mixture was thenallowed to react at room temperature with gentle agitation for 2 hrs. Toobtain a 1:1 complex of PEG and interferon alpha (IFNα-PEG complex),where PEG was linked selectively to the amino group of a lysine residueof interferon alpha, the reaction mixture was subjected to Superdex sizeexclusion chromatography. The IFNα-PEG complex was eluted from thecolumn using 10 mM potassium phosphate buffer (pH 6.0) as an elutionbuffer, and interferon alpha not linked to PEG, unreacted PEG and dimerbyproducts in which two interferon alpha molecules were linked to bothends of PEG were removed. To link the IFNα-PEG complex to the aminegroup of a lysine residue of immunoglobulin Fc, the purified IFNα-PEGcomplex was concentrated to about 5 mg/ml, and an IFNα-PEG-Fc conjugatewas prepared and purified according to the same methods as in the steps3 and 4 of Example 5. Through this experiment, the optimal reactionmolar ratio for IFNα to PEG, providing the highest reactivity andgenerating the fewest byproducts such as dimers, was found to be 1:2.5to 1:5.

On the other hand, another IFNα-PEG-Fc conjugate was prepared accordingto the same methods as described above using PEG) having anN-hydroxysuccinimidyl (NHS) reactive group at both ends, NHS-PEG-NHS(Shearwater), or PEG having a butyl aldehyde reactive group at bothends, BUA-PEG-BUA (Shearwater).

Example 11 Preparation of Protein Conjugate Using PEG Having DifferentMolecular Weight

An IFNα-10K PEG complex was prepared using 10-kDa polyethylene glycolhaving an aldehyde reactive group at both ends, ALD-PEG-ALD(Shearwater). This complex was prepared and purified according to thesame method as in the step 2 of Example 5. Through this experiment, theoptimal reaction molar ratio for IFNα to 10-kDa PEG, providing thehighest reactivity and generating the fewest byproducts such as dimers,was found to be 1:2.5 to 1:5. The purified IFNα-10K PEG complex wasconcentrated to about 5 mg/ml, and, using this concentrate, an IFNα-10KPEG-Fc conjugate was prepared and purified according to the same methodsas in the steps 3 and 4 of Example 5.

Example 12 Preparation of Fab′-S-PEG-N-Fc Conjugate (—SH Group)

<Step 1> Expression and Purification of Fab′

An E. coli transformant, BL21/poDLHF (accession number: KCCM-10511),expressing anti-tumor necrosis factor-alpha Fab′, was grown in 100 ml ofLB medium overnight with agitation, and was inoculated in a 5-Lfermentor (Marubishi) and cultured at 30° C. and 500 rpm and at an airflow rate of 20 vvm. To compensate for the insufficient nutrients forbacterial growth during fermentation, the cultures were supplementedwith glucose and yeast extracts according to the fermented states ofbacteria. When the cultures reached an OD₆₀₀ value of 80-100, aninducer, IPTG, was added to the cultures to induce protein expression.The cultures were further cultured for 40 to 45 hrs until the OD valueat 600 nm increased to 120 to 140. The fermentation fluid thus obtainedwas centrifuged at 20,000×g for 30 min. The supernatant was collected,and the pellet was discarded.

The supernatant was subjected to the following three-step columnchromatography to purify anti-tumor necrosis factor-alpha Fab′. Thesupernatant was loaded onto a HiTrap protein G column (5 ml, Pharmacia)equilibrated with 20 mM phosphate buffer (pH 7.0), and the column waseluted with 100 mM glycine (pH 3.0). The collected Fab′ fractions werethen loaded onto a Superdex 200 column (Pharmacia) equilibrated with 10mM sodium phosphate buffer (PBS, pH 7.3), and this column was elutedwith the same buffer. Finally, the second Fab′ fractions were loadedonto a polyCAT 21×250 column (PolyLC), and this column was eluted with alinear NaCl gradient of 0.15-0.4 M in 10 mM acetate buffer (pH 4.5),thus providing highly pure anti-tumor necrosis factor-alpha Fab′fractions.

<Step 2> Preparation and Purification of Fc-PEG Complex

To link a PEG linker to the N-terminus of an immunoglobulin Fc, theimmunoglobulin Fc prepared according to the same method as in the step 1of Example 5 was dissolved in 100 mM phosphate buffer (pH 6.0) at aconcentration of 5 mg/ml, and was mixed with NHS-PEG-MAL (3.4 kDa,Shearwater) at an Fc:PEG molar ratio of 1:10, followed by incubation 4°C. for 12 hrs with gentle agitation.

After the reaction was completed, the reaction buffer was exchanged with20 mM sodium phosphate buffer (pH 6.0) to remove unbound NHS-PEG-MAL.Then, the reaction mixture was loaded onto a polyCAT column (PolyLC).The column was eluted with a linear NaCl gradient of 0.15-0.5 M in 20 mMsodium phosphate buffer (pH 6.0). During this elution, theimmunoglobulin Fc-PEG complex was eluted earlier than unreactedimmunoglobulin Fc, and the unreacted Ig Fc was eluted later, therebyeliminating the unreacted Ig Fc molecules.

<Step 3> Preparation and Purification of Fab′-S-PEG-N-Fc Conjugate (—SHGroup)

To link the immunoglobulin Fc-PEG complex to a cysteine group of theFab′, the Fab′ purified in the above step 1 was dissolved in 100 mMsodium phosphate buffer (pH 7.3) at a concentration of 2 mg/ml, and wasmixed with the immunoglobulin Fc-PEG complex prepared in the above step2 at a Fab′:complex molar ratio of 1:5. The reaction mixture wasconcentrated to a final protein concentration of 50 mg/ml and incubatedat 4° C. for 24 hrs with gentle agitation.

After the reaction was completed, the reaction mixture was loaded onto aSuperdex 200 column (Pharmacia) equilibrated with 10 mM sodium phosphatebuffer (pH 7.3), and the column was eluted with the same buffer at aflow rate of 1 ml/min. The coupled Fab′-S-PEG-N-Fc conjugate was elutedrelatively earlier due to its high molecular weight, and unreactedimmunoglobulin Fc-PEG complex and Fab′ were eluted later, therebyeliminating the unreacted molecules. To completely eliminate unreactedimmunoglobulin Fc-PEG, the collected Fab′-S-PEG-N-Fc conjugate fractionswere again loaded onto a polyCAT 21×250 column (PolyLC), and this columnwas eluted with a linear NaCl gradient of 0.15-0.5 M in 20 mM sodiumphosphate buffer (pH 6.0), thus providing a pure Fab′-S-PEG-N-Fcconjugate comprising the Fc-PEG complex linked to an —SH group near theC-terminus of the Fab′.

Example 13 Preparation of Fab′-N-PEG-N-Fc Conjugate (N-Terminus)

<Step 1> Preparation and Purification of Fab′-PEG Complex (N-Terminus)

40 mg of the Fab′ purified in the step 1 of Example 12 was dissolved in100 mM sodium phosphate buffer (pH 6.0) at a concentration of 5 mg/ml,and was mixed with butyl ALD-PEG-butyl ALD (3.4 kDa, Nektar) at aFab′:PEG molar ratio of 1:5. A reducing agent, NaCNBH₃, was added to thereaction mixture at a final concentration of 20 mM, and the reactionmixture was then allowed to react at 4° C. for 2 hrs with gentleagitation.

After the reaction was completed, the reaction buffer was exchanged with20 mM sodium phosphate buffer (pH 6.0). Then, the reaction mixture wasloaded onto a polyCAT column (PolyLC). The column was eluted with alinear NaCl gradient of 0.15-0.4 M in 20 mM acetate buffer (pH 4.5).During this column elution, the Fab′-PEG complex comprising the PEGlinker lined to the N-terminus of the Fab′ was eluted earlier thanunreacted Fab′, and the unreacted Fab′ was eluted later, therebyeliminating the unreacted Fab′ molecules.

<Step 2> Preparation and Purification of Fab′-N-PEG-N-Fc Conjugate

To link the Fab′-PEG complex purified in the above step 1 to theN-terminus of an immunoglobulin Fc, the Fab′-PEG complex was dissolvedin 100 mM sodium phosphate buffer (pH 6.0) at a concentration of 10mg/ml, and was mixed with the immunoglobulin Fc dissolved in the samebuffer at a Fab′-PEG complex:Fc molar ratio of 1:5. After the reactionmixture was concentrated to a final protein concentration of 50 mg/ml, areducing agent, NaCNBH₃, was added to the reaction mixture at a finalconcentration of 20 mM, and the reaction mixture was then reacted at 4°C. for 24 hrs with gentle agitation.

After the reaction was completed, the reaction mixture was loaded onto aSuperdex 200 column (Pharmacia) equilibrated with 10 mM sodium phosphatebuffer (pH 7.3), and the column was eluted with the same buffer at aflow rate of 1 ml/min. The coupled Fab′-N-PEG-N-Fc conjugate was elutedrelatively earlier due to its high molecular weight, and unreactedimmunoglobulin Fc and Fab′-PEG complex were eluted later, therebyeliminating the unreacted molecules. To completely eliminate theunreacted immunoglobulin Fc molecules, the collected Fab′-N-PEG-N-Fcconjugate fractions were again loaded onto a polyCAT 21×250 column(PolyLC), and this column was eluted with a linear NaCl gradient of0.15-0.5 M in 20 mM sodium phosphate buffer (pH 6.0), thus providing apure Fab′-N-PEG-N-Fc conjugate comprising the immunoglobulin Fc-PEGcomplex linked to the N-terminus of the Fab′.

Example 14 Preparation and Purification of Deglycosylated ImmunoglobulinFc

200 mg of an immunoglobulin Fc prepared according to the same method asin Example 5 was dissolved in 100 mM phosphate buffer (pH 7.5) at aconcentration of 2 mg/ml, and was mixed with 300 U/mg of adeglycosylase, PNGase F (NEB). The reaction mixture was allowed to reactat 37° C. for 24 hrs with gentle agitation. Then, to purify thedeglycosylated immunoglobulin Fc, the reaction mixture was loaded onto aSP Sepharose FF column (Pharmacia), and the column was eluted with alinear NaCl gradient of 0.1-0.6 M in 10 mM acetate buffer (pH 4.5) using1 M NaCl. The native immunoglobulin Fc was eluted earlier, and thedeglycosylated immunoglobulin Fc (DG Fc) was eluted later.

Example 15 Preparation of IFNα-PEG-DG Fc Conjugate

To link the deglycosylated immunoglobulin Fc prepared in Example 14 tothe IFNα-PEG complex purified in the step 2 Example 5, the IFNα-PEGcomplex was mixed with the DG Fc dissolved in 10 mM phosphate buffer atIFNα-PEG complex:DF Fc molar ratios of 1:1, 1:2, 1:4 and 1:8. After thephosphate buffer concentration of the reaction solution was adjusted to100 mM, a reducing agent, NaCNBH₃, was added to the reaction solution ata final concentration of 20 mM and was allowed to react 4° C. for 20 hrswith gentle agitation. Through this experiment, the optimal reactionmolar ratio for IFNα-PEG complex to DG Fc, providing the highestreactivity and generating the fewest byproducts such as dimers, wasfound to be 1:2.

After the coupling reaction, the reaction mixture was subjected to sizeexclusion chromatography using a Superdex® column (Pharmacia) so as toeliminate unreacted substances and byproducts and purify the IFNα-PEG-DGFc protein conjugate. After the reaction mixture was loaded onto thecolumn, a phosphate buffer (pH 7.3) was passed through the column at aflow rate of 2.5 ml/min to remove unbound DG Fc and unreactedsubstances, followed by column elution to collect IFNα-PEG-DG Fc proteinconjugate fractions. Since the collected IFNα-PEG-DG Fc proteinconjugate fractions contained a small amount of impurities, unreacted DGFc and IFNα-PEG complex, cation exchange chromatography was carried outto remove the impurities. The IFNα-PEG-DG Fc protein conjugate fractionswere loaded onto a PolyCAT LP column (PolyLC) equilibrated with 10 mMsodium acetate (pH 4.5), and the column was eluted with a lineargradient of 0-0.6 M NaCl in 10 mM sodium acetate buffer (pH 4.5) using 1M NaCl. Finally, the IFNα-PEG-DG Fc protein conjugate was purified usingan anion exchange column. The IFNα-PEG-Fc protein conjugate fractionswere loaded onto a PolyWAX LP column (PolyLC) equilibrated with 10 mMTris-HCl (pH 7.5), and the column was then eluted with a linear gradientof 0-0.3 M NaCl in 10 mM Tris-HCl (pH 7.5) using 1 M NaCl, thusisolating the IFNα-PEG-DG Fc protein conjugate in a highly pure form.

Example 16 Preparation of Conjugate of IFNα-PEG Complex and RecombinantAG Fc Derivative

According to the same methods as in Examples 5 and 15, the IFNα-PEGcomplex was linked to the N terminus of the IgG4 delta-Cys as an AG Fcderivative prepared in Example 1. After the coupling reaction, unreactedsubstances and byproducts were removed from the reaction mixture, andthe thus-produced IFNα-PEG-AG Fc protein conjugate (I) was primarilypurified using 50 ml of a Q HP 26/10 column (Pharmacia) and furtherpurified by a high-pressure liquid chromatographic assay using a polyCAT21.5×250 column (polyLC), thus purifying the conjugate to a high degree.The coupling reaction solution was desalted using a HiPrep 26/10desalting column (Pharmacia) with 10 mM Tris buffer (pH 8.0). Then, thereaction solution was then loaded onto 50 ml of a Q HP 26/10 column(Pharmacia) at a flow rate of 8 ml/min, and this column was eluted witha linear NaCl gradient of 0-0.2 M to obtain desired fractions. Thecollected fractions were again loaded onto a polyCAT 21.5×250 columnequilibrated with 10 mM acetate buffer (pH 5.2) at a flow rate of 15ml/min, and this column was eluted with a linear NaCl gradient of0.1-0.3 M, thus providing highly pure fractions. According to the samemethod as described above, another IFNα-PEG-AG Fc protein conjugate (II)was prepared using another AG Fc derivative prepared in Example 1-2,IgG4 monomer.

Example 17 Preparation of EPO-PEG-Recombinant AG Fc Derivative Conjugate

According to the same method as in Example 16, an EPO-PEG-recombinant AGFc derivative conjugate was prepared by linking an AG Fc derivative,IgG4 delta-Cys, to the EPO-PEG complex.

Comparative Example 1 Preparation of IFNα-40K PEG Complex

5 mg of interferon alpha was dissolved in 100 mM phosphate buffer toobtain a final volume of 5 ml, and was mixed with 40-kDa activatedmethoxy PEG-aldehyde (Shearwater), at an IFNα:40-kDa PEG molar ratio of1:4. To this mixture, a reducing agent, NaCNBH₃ was added at a finalconcentration of 20 mM and was allowed to react at 4° C. for 18 hrs withgentle agitation. To inactivate PEG, which did not react with IFNα,Ethanolamine was added to the reaction mixture at a final concentrationof 50 mM.

A Sephadex G-25 column (Pharmacia) was used to remove unreacted PEG andexchange the buffer with another buffer. First, this column wasequilibrated with two column volumes (CV) of 10 mM Tris-HCl buffer (pH7.5), and was loaded with the reaction mixture. Flow throughs weredetected by measuring the absorbance at 260 nm using a UVspectrophotometer. When the column was eluted with the same buffer,interferon alpha modified by adding PEG having a higher molecular weightto its N-terminus was eluted earlier, and unreacted PEG as eluted later,thus allowing isolation of only IFNα-40K PEG.

The following chromatography was carried out to further purify theIFNα-40K PEG complex from the collected fractions. 3 ml of a PolyWAX LPcolumn (PolyLC) was equilibrated with 10 mM Tris-HCl (pH 7.5). Thecollected fractions containing the IFNα-40K PEG complex was loaded ontocolumn at a flow rate of 1 ml/min, and the column was washed with 15 mlof the equilibrium buffer. Then, the column was eluted with a linearNaCl gradient of 0-100% using 30 ml of 1 M NaCl thus eluting interferonalpha conjugated to tri-, di- and mono-PEG, sequentially. To furtherpurify the mono-PEG-conjugated interferon alpha, the collected fractionscontaining the mono-PEG-conjugated interferon alpha were subjected tosize exclusion chromatography. The fractions were concentrated andloaded onto a Superdex 200 column (Pharmacia) equilibrated with 10 mMsodium phosphate buffer (pH 7.0), and the column was eluted with thesame buffer at a flow rate of 1 ml/min. The tri- and di-PEG-conjugatedinterferon alpha molecules were removed based on their property of beingeluted earlier than the mono-PEG-conjugated interferon alpha, thusisolating the mono-PEG-conjugated interferon alpha in a highly pureform.

According to the same method as described above, 40-kDa PEG wasconjugated to the N-terminus of human growth hormone, granulocyte colonystimulating factor (G-CSF), and a derivative of G-CSF, thus providinghGH-40K PEG, G-CSF-40K PEG and 40K PEG-¹⁷S-G-CSF derivative complexes.

Comparative Example 2 Preparation of IFNα-PEG-Albumin Conjugate

To link the IFNα-PEG complex purified in the step 2 of Example 1 to theN-terminus of albumin, the IFNα-PEG complex was mixed with human serumalbumin (HSA, about 67 kDa, Green Cross) dissolved in 10 mM phosphatebuffer at an IFNα-PEG complex:albumin molar ratio of 1:1, 1:2, 1:4 and1.8. After the phosphate buffer concentration of the reaction solutionwas adjusted to 100 mM, a reducing agent, NaCNBH₃, was added to thereaction solution at a final concentration of 20 mM and was allowed toreact at 4° C. for 20 hrs with gentle agitation. Through thisexperiment, the optimal reaction molar ratio for IFNα-PEG complex toalbumin, providing the highest reactivity and generating the fewestbyproducts such as dimers, was found to be 1:2.

After the coupling reaction, the reaction mixture was subjected to sizeexclusion chromatography using a Superdex® column (Pharmacia) so as toeliminate unreacted substances and byproducts and purify theIFNα-PEG-albumin protein conjugate produced. After the reaction mixturewas concentrated and loaded onto the column, 10 mM sodium acetate bufferpassed through the column at a flow rate of 2.5 ml/min to remove unboundalbumin and unreacted substances, followed by column elution to purifyonly IFNα-PEG-albumin protein conjugate. Since the collectedIFNα-PEG-albumin protein conjugate fractions contained a small amount ofimpurities, unreacted albumin and interferon alpha dimers, cationexchange chromatography was carried out to remove the impurities. TheIFNα-PEG-albumin protein conjugate fractions were loaded onto SP5PWcolumn (Waters) equilibrated with 10 mM sodium (pH 4.5), and the columnwas eluted with a linear gradient of 0-0.5 M NaCl in 10 mM sodiumacetate buffer (pH 4.5) using 1 M NaCl, thus isolating theIFNα-PEG-albumin protein conjugate in a highly pure form.

According to the sane method as described above, albumin was conjugatedto human growth hormone, G-CSF, and a derivative of G-CSF, thusproviding hGH-PEG-albumin, G-CSF-PEG-albumin and ¹⁷S-G-CSF-PEG-albuminconjugates.

Comparative Example 3 Preparation of Fab′-S-40K PEG Complex

The free cysteine residue of the Fab′ purified in the step 1 of Example8 was activated by incubation in an activation buffer (20 M acetate (pH4.0), 0.2 mM DTT) for 1 hr. After the buffer was exchanged by a PEGmodification buffer, 50 mM potassium phosphate (pH 6.5), maleimide-PEG(MW: 40 kDa, Shearwater) as added thereto at a Fab′:40-kDa PEG molarratio of 1:10 and was reacted to react at 4° C. for 24 hrs with gentleagitation.

After the reaction was completed, the reaction solution was loaded ontoa Superdex 200 column (Pharmacia) equilibrated with 10 mM sodiumphosphate buffer (pH 7.3), and the column was eluted with the samebuffer at a flow rate of 1 ml/ml. The Fab′ conjugated 40-kDa PEG(Fab′-40K PEG) was eluted relatively earlier due to its high molecularweight, and unreacted Fab′ was eluted later, thereby eliminating theunreacted Fab′. To completely eliminate the unreacted Fab′, thecollected Fab′-40K PEG complex fractions were again loaded onto apolyCAT 21×250 column (PolyLC), and this column was eluted with a linearNaCl gradient of 0.15-0.5 M in 20 mM sodium phosphate buffer (pH 4.5),thus providing a pure Fab′-S-40K PEG complex comprising 40-kDa PEGlinked to an —SH group of the Fab′.

Experimental Example 1 Identification and Quantitative Analysis of theProtein Conjugates

<1-1> Identification of the Protein Conjugate

The protein conjugates prepared in the above Examples were analyzed bynon-reduced SDS-PAGE using a 4-20% gradient gel and a 12% gel and ELISA(R&D system). As a result of SDS-PAGE analysis, as shown in FIG. 6, acoupling reaction of a physiological polypeptide, a non-peptide polymer,PEG, and an immunoglobulin Fc fragment resulted in the successfulproduction of an IFNα-PEG-Fc conjugate (A), a ¹⁷Ser-G-CSF-PEG-Fcconjugate (B) and an EPO-PEG-Fc conjugate (C).

In addition, the DG Fc prepared in Example 10 was analyzed bynon-reduced 12% SDS-PAGE. As shown in FIG. 9b , a DG Fc band wasdetected at a position, which corresponds to the molecular weight of thenative Fc lacking sugar moieties.

<1-2> Quantitative Analysis of the Protein Conjugates

The protein conjugates prepared in the above Examples were quantified bysize exclusion chromatography using a HiLoad 26/60 Superdex 75 column(Pharmacia) and 10 mM potassium phosphate buffer (pH 6.0) as an elutionbuffer, wherein a peak area of each protein conjugate was compared tothat of a control group. Previously quantitatively analyzed standards,IFNα, hGH, G-CSF, ¹⁷S-G-CSF, EPO and Fc, were individually subjected tosize exclusion chromatography, and a conversion factor between aconcentration and a peak was determined. A predetermined amount of eachprotein conjugate was subjected to the same size exclusionchromatography. By subtracting a peak area corresponding to animmunoglobulin Fc fragment from the thus-obtained peak area, aquantitative value for a physiologically active protein present in eachprotein conjugate was determined. FIG. 7 shows the result of sizeexclusion chromatography of the purified IFNα-PEG-Fc conjugate, whereina single peak was observed. This result indicates that the purifiedprotein conjugate does not contain multimeric impurities such as adimer, a trimer or a higher number of monomers.

When a physiologically active polypeptide conjugated to Fc wasquantitatively analyzed using an antibody specific to thephysiologically active polypeptide, the antibody was prevented frombinding to the polypeptide, resulting in a value lower than an actualvalue calculated by the chromatography. In the case of the IFNα-PEG-Fcconjugate, an ELISA resulted in an ELISA value corresponding to about30% of an actual value.

<1-3> Evaluation of Purity and Mass of the Protein Conjugates

The protein conjugates prepared in the above Examples were subjected tosize exclusion chromatography, and absorbance was measured at 280 nm. Asa result, the IFNα-PEG-Fc, hGH-PEG-Fc, G-CSF-PEG-Fc and¹⁷Ser-G-CSF-PEG-Fc conjugates displayed a single peak at the retentiontime of a 70 to 80-kDa substance.

On the other hand, reverse phase HPLC was carried out to determinepurities of the protein conjugates prepared in Examples 5, 15 and 16,IFNα-PEG-Fc, IFNα-PEG-DG Fc and IFNα-PEG-recombinant AG Fc derivative. Areverse phase column (259 VHP54 column, Vydac) was used. The column waseluted with a 40-100% acetonitrile gradient with 0.5% TFA, and puritieswere analyzed by measuring absorbance at 280 nm. As a result, as shownin FIG. 11, the samples contain no unbound interferon or immunoglobulinFc, and all of the protein conjugates, IFNα-PEG-Fc (A), IFNα-PEG-DG Fc(B) and IFNα-PEG-recombinant AG Fc derivative (C), were found to have apurity greater than 96%.

To determine accurate molecular weights of the purified proteinconjugates, mass for each conjugate was analyzed using a high-throughputMALDI-TOF mass spectrophotometer (Voyager DE-STR, Applied Biosystems).Sinapinic acid was used as a matrix. 0.5 μl of each test sample wascoated onto a sample slide and air-dried, again mixed with the equalvolume of a matrix solution and air-dried, and introduced into an ionsource. Detection was carried out in a positive fashion using a linearmode TOF analyzer. Ions were accelerated with a split extraction sourceoperated with delayed extraction (DE) using a delayed extraction time of750 nsec to 1500 nsec at a total acceleration voltage of about 2.5 kV.

The molecular weights observed by MALDI-TOF mass spectrometry for the Fcprotein conjugates prepared in Examples are given in Table 1, below.FIG. 8 shows the result of MALDI-TOF mass spectrometry of the EPO-PEG-Fcconjugate, and FIG. 10 shews the results of MALDI-TOF mass spectrometryof the IFNα-PEG-Fc and IFNα-PEG-DG Fc conjugates. As a result, theEPO-PEG-Fc protein conjugate was found to have a purity of more than 95%and a molecular weight very close to a theoretical MW. Also, EPO wasfound to couple to the immunoglobulin Fc fragment at a ratio of 1:1.

TABLE 1 Theoretical Measured MW (kDa) MW (kDa) IFNα-PEG-Fc (E.1) 75.475.9 hGH-PEG-Fc (E.3) 78.4 78.6 G-CSF-PEG-Fc (E.4) 75.3 75.9 ¹⁷S-G-CSFderivative-PEG-Fc (E.4) 75.0 75.9 EPO-PEG-Fc (E.5) 91.4 91.0

In addition, when the Fc and DG Fc prepared in Example 14 were examinedfor their molecular weights by MALDI-TOF mass spectrometry, the DS Fcwas found to be 50 kDa, which is about 3-kDa less than native Fc (FIG.9a ). Since the 3-kDa MW corresponds to the theoretical size of sugarmoieties, the results demonstrate that the sugar moieties are completelyremoved.

Table 2, below, shows the results of MALDI-TOF mass spectrometry of theIFNα-PEG-DG Fc conjugate prepared in Example 11 and theIFNα-PEG-recombinant AG Fc derivative conjugates (I and II) prepared inExample 16. The IFNα-PEG-DG Fc conjugate was found to be 3 kDa lighter,and the IFNα-PEG-recombinant AG Fc derivative conjugate (I) to be about3-4 kDa lighter, than the IFNα-PEG-Fc conjugate of 75.9 kDa. TheIFNα-PEG-recombinant AG Fc derivative conjugate (II) coupled to an Fcmonomer showed a molecular weight decreased by 24.5 kDa corresponding tothe molecular weight of the Fc monomer.

TABLE 2 Theoretical Measured MW (kDa) MW (kDa) IFNα-PEG-DG Fc (E.11)72.8 73.0 IFNα-PEG-recombinant AG Fc 72.3 72.2 derivative (I) (E.13)IFNα-PEG-recombinant AG Fc 46.8 46.6 derivative (II) (E.13)

Experimental Example 2 Pharmacokinetic Analysis I

Native forms of physiologically active proteins (controls) and theprotein complexes prepared in Examples and Comparative Examples, -40KPEG complexes, -PEG-albumin conjugates, -PEG-Fc conjugates, -PEG-DG Fcconjugates and -PEG-recombinant AG Fc derivative conjugates, wereevaluated for serum stability and pharmacokinetic parameters in SD rats(five rats per group). The controls, and the -40K PEG complexes,-PEG-albumin conjugates, -PEG-Fc conjugates, -PEG-DG Fc conjugates and-PEG-recombinant AG Fc derivative conjugates (test groups) wereindividually injected subcutaneously at a dose of 100 μg/kg. After thesubcutaneous injection, blood samples were collected at 0.5, 1, 2, 4, 6,12, 24, 30, 48, 72 and 96 hrs in the control groups, and, in the testgroups, at 1, 6, 12, 24, 30, 48, 72, 96, 120, 240 and 288 hrs. The bloodsamples were collected in tubes with an anticoagulant, heparin, andcentrifuged for 5 min using an Eppendorf high-speed micro centrifugatorto remove blood cells. Serum protein levels were measured by ELISA usingantibodies specific to the physiologically active proteins.

The results of pharmacokinetic analyses of the native forms of IFNα,hGH, G-CSF and EPO, and -40K PEG complexes thereof, -PEG-albuminconjugates thereof, -PEG-Fc conjugates thereof and -PEG-DG Fc conjugatesthereof, are given in Tables 3 to 7, below. In the following tables,T_(max) indicates the time taken to reach the maximal drug serumconcentration, T_(1/2) indicates the serum half-life of a drug, and MRT(mean residence time) indicates the mean time that a drug moleculeresides in the body.

TABLE 3 Pharmacokinetics of interferon alpha IFNα-PEG- IFNα-PEG- IFNα-recombinant recombinant IFNα- PEG- IFNα- IFNα-PEG- AG Fc AG Fc Native40K PEG albumin PEG-Fc DG Fc derivative derivative IFNα (C.E. 1) (C.E.2) (E. 5) (E. 15) (I) (E. 16) (II) (E. 16) T_(max) (hr) 1.0 30 12 30 4824 24 T_(1/2) (hr) 1.7 35.8 17.1 90.4 71.0 61.2 31.2 MRT (hr) 2.1 71.532.5 150.1 120.6 111.0 58.8

TABLE 4 Pharmacokinetics of human growth factor hGH-PEG- Native hGH-40KPEG albumin hGH-PEG-Fc hGH (C.E.1) (C.E.2) (E.7) T_(max) (hr) 1.0 12 1212 T_(1/2) (hr) 1.1 7.7 5.9 11.8 MRT (hr) 2.1 18.2 13.0 18.8

TABLE 5 Pharmacokinetics of G-CSF G-CSF-PEG- Native G-CSF-40K PEGalbumin G-CSF-PEG-Fc G-CSF (C.E.1) (C.E.2) (E.8) T_(max) (hr) 2.0 12 1212 T_(1/2) (hr) 2.8 4.8 5.2 6.9 MRT (hr) 5.2 24.5 25.0 32.6

TABLE 6 Pharmacokinetics of ¹⁷S-G-CSF derivative Native ¹⁷S-G-CSF-¹⁷S-G-CSF-PEG- ¹⁷S-G-CSF- ¹⁷S-G-CSF 40K PEG albumin PEG-Fc derivative(C.E.1) (C.E.2) (E.8) T_(max) (hr) 2.0 24 24 24 T_(1/2) (hr) 2.9 4.3 6.47.0 MRT (hr) 5.8 24.4 25.1 33.2

TABLE 7 Pharmacokinetics of EPO EPO-PEG- recombinant Highly AG Fc Nativeglycosylated EPO-PEG-Fc derivative EPO EPO (E.9) (E.17) T_(max) (hr) 6.012 30 48 T_(1/2) (hr) 9.4 18.4 61.5 87.9 MRT (hr) 21.7 26.8 117.6 141.6

As shown from the data of Table 13 and the pharmacokinetic graph of FIG.12, the IFNα-PEG-Fc protein conjugate had a serum half-life of 90.4 hrs,which was about 50 times higher than that of native IFNα and about 2.5times higher than that of IFNα-40K PEG having a half-life of 35.8 hrs,prepared in Comparative Example 1. Also, the IFNα-PEG-Fc proteinconjugate of the present invention was found to be superior in serumhalf-life to IFNα-PEG-albumin, which has a half-life of 17.1 hrs.

On the other hand, as shown in Table 3 and FIG. 14, the IFNα-PEG-DG Fcconjugate had a serum half-life of 71.0 hrs, which was almost the sameas the IFNα-PEG-Fc conjugate, indicating that the deglycosylation of Fcdoes not greatly affect the in vivo stability of the IFNα-PEG-DG Fcconjugate. Also, the conjugate prepared using the recombinant AG Fcderivative produced by a recombinant method was found to have an effectidentical to that of the native form-derived DG Fc. However, the serumhalf-life of a complex coupled to an Fc monomer was about half that of acomplex coupled to a normal Fc dimer.

As shown in Table 4, human growth hormone also showed an extended serumhalf-life when conjugated to the IgG Fc fragment according to thepresent invention. That is, compared to the native form (1.1 hrs), thehGH-40K PEG complex and hGH-PEG-albumin conjugate had slightly increasedhalf-lives of 7.7 hrs and 5.9 hrs, respectively, whereas the hGH-PEG-Fcprotein conjugate of the present invention displayed a greatly extendedserum half-life of 11.8 hrs.

As apparent from the pharmacokinetic data of G-CSF and its derivative inTable 5 and 6, the G-CSF-PEG-Fc and ¹⁷S-G-CSF-PEG-Fc conjugatesdisplayed a much longer serum half-life than the -40K PEG complex and-PEG-albumin conjugate. The immunoglobulin Fc fragment was found in theserum to prolong the duration of action of physiologically activeproteins in native forms, as well as in their derivatives havingalterations of certain amino acid residues in similar levels to thenative forms. From these results, it is easily predictable that themethod of the present invention will have a similar effect on otherproteins and their derivatives.

As shown in Table 7 and FIG. 13, the conjugation of the nativeglycosylated EPO to the Fc fragment also resulted in an increase inserum half-life. That is, EPO had a serum half-life of 9.4 hrs in thenative form, and a prolonged serum half-life of 18.4 hrs in theDarbepoetin α(Aranesp, Amgen), which is highly glycosylated to improveserum stability. The EPO-PEG-Fc conjugate, comprising EPO coupled to theimmunoglobulin Fc fragment according to the present invention, displayeda markedly prolonged serum half-life of 61.5 hrs. Also, when conjugatedto the E. coli-derived recombinant aglycosylated (AG) Fc derivative, thehalf-life of EPO increased to 87.9 hrs, indicating that theaglycosylation of the Fc fragment allows the preparation of a proteinconjugate not affecting serum stability of the protein without antibodyfunctions.

As apparent from the above results, the protein conjugatescovalent-bonded to the immunoglobulin Fc fragment through a non-peptidepolymer according to the present invention displayed serum half-livesincreased several to several tens to that of the native form. Also, whenthe immunoglobulin Fc was aglycosylated by production in E. coli ordeglycosylated by enzyme treatment, its effect of increasing the serumhalf-life of its protein conjugate was maintained at a similar level.

In particular, compared to proteins modified with 40-kDa PEG having thelongest duration of action among PEG molecules for increasing theduration of action of proteins in the serum, the immunoglobulin Fcprotein conjugates had much superior serum stability. In addition,compared to protein conjugates coupled to albumin instead of theimmunoglobulin Fc, the protein conjugates of the present inventiondisplayed excellent serum stability, indicating that the proteinconjugates of the present invention are effective in developinglong-acting forms of protein drugs. These results, that the presentprotein conjugates have excellent effects on serum stability and MRT ina broad range of proteins including colony stimulating factorderivatives by point mutation compared to conventional PEG- oralbumin-conjugated proteins, indicate that the stability andduration-extending effects of the present protein conjugates areapplicable to other physiologically active polypeptides.

On the other hand, when the IFNα-10K PEG-Fc protein conjugate (Example11) prepared using a non-peptide polymer, 10-kDa PEG, was evaluated forits serum half-life according to the same method as described above, itshowed a serum half-life of 48.8 hrs, which was somewhat shorter thanthe serum half-life (79.7 hrs) of a protein conjugate prepared using3.4-kDa PEG.

In addition, the serum half-lives of the protein conjugates decreaseswith increasing molecular weight of the non-peptide polymer PEG. Theseresults indicate that the major factor increasing the serum stabilityand duration of the protein conjugates is the conjugated immunoglobulinFc fragment rather than the non-peptide polymer.

Even when the reactive group of PEG was exchanged with a reactive groupother than the aldehyde group, protein conjugates with the PEG showedsimilar patterns in apparent molecular weight and serum half-life tothose coupled to PEG having an aldehyde reactive group.

Experimental Example 3 Pharmacokinetic Analysis II

To determine the serum half-lives of the Fab′-S-PEG-N-Fc andFab′-N-PEG-N-Fc conjugates prepared in Example 12 and 13 and theFab′-S-40K PEG complex prepared in Comparative Example 3, drugpharmacokinetic analysis was carried out according to the same method asin Experimental Example 2 using Fab′ as a control, the conjugates andthe complex. The results are given in FIG. 15.

As shown in FIG. 15, the Fab′-S-PEG-N-Fc and Fab′-N-PEG-N-Fc conjugatesdisplayed a serum half-life prolonged two or three times compared to theFab′ or Fab′-S-40K PEG complex.

Experimental Example 4 Evaluation of Intracellular Activity of theProtein Conjugates

<4-1> Comparison of the IFNα Protein Conjugates for IntracellularActivity

To compare the intracellular activity of the IFNα protein conjugates,the IFNα-PEG-Fc (Example 5), IFNα-PEG-DG Fc (Example 15),IFNα-PEG-recombinant AG Fc derivative (Example 16), IFNα-40K PEG(Comparative Example 1) and IFNα-PEG-albumin (Comparative Example 2)were evaluated for antiviral activity by a cell culture bioassay usingMadin Darby Bovine Kidney (MDBK) cells (ATCC CCL-22) infected withvesicular stomatitis virus. Nonpegylated interferon alpha-2b, availablefrom the National Institute for Biological Standards and Controls(NIBSC), was used as a standard material.

MDBK cells were cultured in MEM (minimum essential medium, JBI)supplemented with 10% FBS and 1% penicillin/streptomycin at 37° C. under5% CO₂ condition. Samples to be analyzed and the standard material werediluted with the culture medium to predetermined concentrations, and100-μl aliquots were placed onto each well of a 96-well plate. Thecultured cells were detached, added to the plate containing the samplesin a volume of 100 μl, and cultured for about 1 hr at 37° C. under 5%CO₂ condition. Then, 50 μl of vesicular stomatitis virus (VSV) of5-7×10³ PFU was added to each well of the plate, and the cells werefurther cultured for about 16 to 20 hrs at 37° C. under 5% CO₂conditions. A well that did not contain the sample or standard materialbut contained only the virus was used as a negative control, and a wellthat contained only cells was used as a positive control.

After the culture medium was removed, 100 μl of a neutral red solutionwas added to the plate to stain viable cells, followed by incubation for2 hrs at 37° C. under 5% CO₂ condition. After the supernatants wereremoved, 100 μl of a 1:1 mixture of 100% ethanol and 1% acetic acid wasadded to each well of the plate. After thorough mixing to dissolve allneutral red crystals eluted from stained cells, absorbance was measuredat 540 nm. The negative control was used as a blank, and ED₅₀ values(doses causing 50% cell growth inhibition) were calculated, where thecell growth of the positive control was set at 100%.

TABLE 8 Specific Relative activity Conc. activity (%) for native (ng/ml)(IU/mg) IFNα Native IFNα 100 4.24E+08 100 IFNα-40K PEG 100 2.04E+07 4.8IFNα-PEG-albumin 100 2.21E+07 5.2 IFNα-PEG-Fc 100 1.19E+08 28.1IFNα-PEG-DG Fc 100 1.09E+08 25.7 IFNα-PEG-recombinant 100 9.58E+07 22.6AG Fc derivative

As shown in Table 8, the IFNα-40K PEG decreased in activity to 4.8% ofthe native IFNα. Especially, as the size of the PEG moieties increased,a protein conjugate has improved serum stability but gradually decreasedactivity. Interferon alpha was reported to have in vitro activities of25% when modified with 12-kDa PEG and about 7% when modified with 40-kDaPEG (P. Bailon et al., Bioconjugate Chem. 12: 195-202, 2001). That is,since a protein conjugate has a longer half-life but sharply decreasesin biological activity as the molecular weight of PEG moieties increase,there is a need for the development of a protein conjugate having alonger serum half-life and a stronger activity. In addition, theIFNα-PEG-albumin conjugate displayed a weak activity of about 5.2%compared to the native IFNα. In contrast, the IFNα-PEG-Fc andIFNα-PEG-DG Fc conjugates of the present invention exhibited a markedlyimproved relative activity of 28.1% and 25.7% compared to the nativeIFNα. Also, the conjugation of IFNα to the recombinant AG Fc derivativeresulted in a similar increase in activity. From these results, it isexpected that interferon alpha conjugated to the immunoglobulin Fcfragment has a markedly increased serum half-life and greatly improvedpharmaceutical efficacy in vivo.

<4-2> Comparison of the Human Growth Hormone-Protein Conjugates forIntracellular Activity

To compare the intracellular activity of the human growth hormoneprotein conjugates, the hGH-PEG-Fc, hGH-40K PEG and hGH-PBG-albumin werecompared for intracellular activity.

Intracellular activities of the hGH conjugates were measured by an invitro assay using a rat node lymphoma cell line, Nb2 (EuropeanCollection of Cell Cultures (ECACC) 190 97041101), which develops humangrowth hormone-dependent mitogenesis.

Nb2 cells were cultured in Fisher's medium supplemented with 10% FBS(fetal bovine serum), 0.075% NaCO₃, 0.05 mM 2-mercaptoethanol and 2 mMglutamin, and were further cultured in a similar medium not containing10% FBS for 24 hrs. Then, the cultured cells were counted, and about2×10⁴ cells were aliquot ted onto each well of a 96-well plate. ThehGH-PEG-Fc, the hGH-40K PEG, the hGH-PEG-albumin, a standard availablefrom the National Institute for Biological Standards and Controls(NIBSC) as a control, and native human growth hormone (HM-hGH) werediluted and added to each well at various concentrations, followed byincubation for 48 hrs at 37° C. under 5% CO₂ condition. Thereafter, tomeasure cell proliferation activity by determining the cell number ineach well, 25 μl of the Cell Titer 96 Aqueous One Solution Reagent(Promega) was added to each well, and the cells were further culturedfor 4 hrs. Absorbance was measured at 490 nm, and a titer for eachsample was calculated. The results are given in Table 9, below.

TABLE 9 Specific Relative activity Conc. activity* (%) for native(ng/ml) (U/mg) HM-hGH Native hGH 100 2.71E+06 100 hGH (standardavailable 100 2.58E+06 95.2 from NIBSC) hGH-40K PEG 100 0.206E+06  7.6hGH-PEG-albumin 100 0.141E+06  5.2 hGH-PEG-Fc 100 0.76E+06 28.1 Specificactivity* = 1/ED₅₀ × 10⁶ (ED₅₀: protein amount required for 50% ofmaximum cell growth

As shown in Table 9, also in the case of human growth hormone, theconjugation to 40-kDa PEG (hGH-40K PEG) resulted in a decrease inactivity to about 7.6% of the native form, and the hGH-PEG-albuminconjugate displayed a low in vitro activity that was about 5.2% of thenative hGH. However, the hGH-PEG-Fc conjugate of the present inventionmarkedly increased in relative activity to greater than 28% compared tothe native hGH. From these results, it is expected that human growthhormone linked to the immunoglobulin Fc fragment has a markedlyincreased serum half-life and a greatly improved in vivo pharmaceuticalefficacy. In addition, it is believed that the increased activity of theimmunoglobulin Fc protein conjugates of the present invention is due tothe increased serum stability and preserved binding affinity toreceptors due to the immunoglobulin Fc or due to the space formed by thenon-peptide polymer. These effects are predicted to be applicable toimmunoglobulin Fc protein conjugates coupled to other physiologicallyactive proteins.

<4-3> Comparison of the G-CSF Protein Conjugates for IntracellularActivity

To compare the intracellular activity of the protein conjugates with aG-CSF derivative, the native G-CSF (Filgrastim, Jeil Pharm. Co., Ltd.),¹⁷Ser-G-CSF derivative, 20K PEG-G-CSF (Neulasta), 40K PEG-¹⁷S-G-CSF,¹⁷Ser-G-CSF-PEG-albumin and ¹⁷S-G-CSF-PEG-Fc were compared forintracellular activity.

First, a human myeloid cell line, HL-60 (ATCC CCL-240, promyelocyticleukemia patient/36 yr old Caucasian female), was cultured in RPMI 1640medium supplemented with 10% FBS. The cultured cells were suspended at adensity of about 2.2×10⁵ cells/ml, and DMSO (dimethylsulfoxide, culturegrade, Sigma) was added thereto at a final concentration of 1.25% (v/v).Then, 90 μl of the cell suspension was seeded onto each well of a96-well plate (Corning/low evaporation 96 well plate), thus providing adensity of about 2×10⁴ cells per well, and cultured in an incubator at37° C. with 5% CO₂ for about 72 hrs.

Each sample, whose protein concentration was determined using a G-CSFELISA kit (R&D systems), was diluted with RPMI 1640 to an identicalconcentration of 10 μg/ml, and further diluted two-fold with RPMI 1640nineteen times. The serial two-fold dilutions were individually added toeach well containing HL-60 cells at a volume of 10 μl, so that theconcentration of each sample started at 1 μg/ml. Then, the cells werecultured in an incubator at 37° C. for 72 hrs.

The proliferation of HL-60 cells was assayed using Cell Titer 96™ (Cat.NO. G4100, Promega), and the increased cell number was determined bymeasuring absorbance at 670 nm.

TABLE 10 ED₅₀ Relative activity (%) (IU/mg) for native G-CSF NativeG-CSF 0.30 100 ¹⁷Ser-G-CSF 0.26 115 G-CSF-20K PEG (Neulasta) 1.20 25¹⁷Ser-G-CSF-40K PEG 10.0 <10.0 ¹⁷Ser-G-CSF-PEG-albumin 1.30 23.0¹⁷Ser-G-CSF-PEG-Fc 0.58 51.7

As shown in Table 10, the immunoglobulin Fc protein conjugates coupledto a G-CSF derivative having an amino acid substitution, ¹⁷Ser-G-CSF,also displayed similar effects to native G-CSF-coupled proteinconjugates. The ¹⁷Ser-G-CSF-PEG was previously reported to have arelatively increased serum half-life but a decreased activity comparedto nonpegylated ¹⁷Ser-G-CSF (Korean Pat. Laid-open Publication No.2004-83268). Especially, as the size of the PEG moieties increased, aprotein conjugate had increased serum stability but gradually decreasedactivity. The ¹⁷Ser-G-CSF-40K PEG showed a very low activity of lessthan about 10% compared to the native form. That is, since a proteinconjugate has an extended serum half-life but a sharply decreasedactivity as the molecular weight of PEG moieties increases, there is theneed for the development of a protein conjugate having a long serumhalf-life and strong activity. The ¹⁷Ser-G-CSF-PEG-albumin also showed alow activity of about 23% compared to the native G-CSF. In contrast, the¹⁷Ser-G-CSF-PEG-Fc was greatly improved in relative activity to morethan 51% compared to the native G-CSF. From these results, it isexpected that ¹⁷Ser-G-CSF linked to the immunoglobulin Fc fragment has amarkedly increased serum half-life and a greatly improved pharmaceuticalin vivo efficacy.

<4-4> Cytotoxicity Neutralization Assay for the Fab′ Conjugates

An in vitro activity assay was carried out using the Fab′-S-PEG-N-Fc andFab′-N-PEG-N-Fc conjugates prepared in Example 8 and 9 and theFab′-S-40K PEG complex prepared in Comparative Example 3. Through acytotoxicity assay based on measuring TNFα-mediated cytotoxicity, theFab′ conjugates were evaluated to determine whether they neutralizeTNFα-induced apoptosis in a mouse fibroblast cell line, L929 (ATCCCRL-2148).

The Fab′-S-PEG-N-Fc and Fab′-N-PEG-N-Fc conjugate and the Fab′-S-40K PEGcomplex were serially two-fold diluted, and 100-μl aliquots were placedonto wells of a 96-well plate. rhTNF-α (R&D systems) and actinomycin D(Sigma) used as an RNA synthesis inhibitor were added to each well atfinal concentrations of 10 ng/ml and 1 μg/ml, respectively, incubatedfor 30 min in an incubator at 37° C. with 5% CO₂, and transferred to amicroplate for assay. L929 cells were added to each well at a density of5×10⁴ cells/50 μl medium and cultured for 24 hrs in an incubator at 37°C. with 5% CO₂. After the culture medium was removed, 50 μl of MTT(Sigma) dissolved in PBS at a concentration of 5 mg/ml was added to eachwell, and the cells were further cultured for about 4 hrs in anincubator at 37° C. with 5% CO₂. 150 μl of DMSO was added to each well,and the degree of cytotoxicity neutralization was determined bymeasuring the absorbance at 540 nm. As a control, the Fab′ purified inthe step 1 of Example 8 was used.

As shown in FIG. 16, all of the protein conjugates used in this test hada similar titer to the Fab′. These results indicate that, when a proteinconjugate is prepared by linking an immunoglobulin Fc to a free cysteineresidue near the N-terminus or C-terminus of a Fab′ through PEG, theFab′ exhibits a markedly increased serum half-life and a high in vivoactivity.

<4-5> Complement-Dependent Cytotoxicity (CDC) Assay

To determine whether the derivatives prepared in Examples and proteinscorresponding to the constant regions of immunoglobulins, expressed inthe E. coli transformants and purified, bind to human C1q, an enzymelinked immunosorbent assay (ELISA) was carried out as follows. As testgroups, immunoglobulin constant regions produced by the HM10932 andHM10927 transformants and the derivatives prepared in the above Exampleswere used. As standards, a glycosylated immunoglobulin (IVIG-globulin S,Green Cross PBM) and several commercially available antibodies used astherapeutic antibodies were used. The test and standard samples wereprepared in 10 mM carbonate buffer (pH 9.6) at a concentration of 1μg/ml. The samples were aliquoted into a 96-well plate (Nunc) in anamount of 200 ng per well, and the plate was coated overnight at 4° C.Then, each well was washed with PBS-T (137 mM NaCl, 2 mM KCl, 10 mMNa₂HPO₄, 2 mM KH₂PO₄, 0.05% Tween 20) three times, blocked with 250 μlof a blocking buffer (1% bovine serum albumin in PBS-T) at roomtemperature for 1 hr, and washed again with the same PBS-T three times.The standard and test samples were diluted in PBS-T to a predeterminedconcentration and added to antibody-coated wells, and the plate wasincubated at room temperature for 1 hr and washed with PBS-T threetimes. Thereafter, 2 μg/ml C1q (R&D Systems) was added to the plate andreacted at room temperature for 2 hrs, and the plate was washed withPBS-T six times. 200 μl of a 1:1000 dilution of a human anti-human C1qantibody-peroxidase conjugate (Biogenesis, USA) in the blocking bufferwas added to each well and reacted at room temperature for 1 hr. Aftereach well was washed with PBS-T three times, equal volumes of colorreagents A and B (Color A: stabilized peroxide and Color B: stabilizedchromogen; DY 999, R&D Systems) were mixed, and 200 μl of the mixturewas added to each well, followed by incubation for 30 min. Then, 50 μlof a reaction termination solution, 2 M sulphuric acid, was added toeach well. The plate was read using a microplate reader (MolecularDevice). Absorbance of standard and test samples was measured at 450 nm,and the results are given in FIGS. 17 and 18, respectively.

When immunoglobulin subclasses were compared with each other forcomplement activity in their immunoglobulin Fc fragment, the highestbinding affinity to C1q was found in human immunoglobulin IgG1(Fitzgerald), the next in IgG2 (Fitzgerald) and then IgG4 (Fitzgerald),indicating that there is a difference between subclasses in complementactivity. The IVIG used in this test, which is a pool of IgG subclasses,exhibited a C1q binding affinity almost the same as the purified IgG1because IgG1 amounts to most of the IVIG. Compared to these standards,with respect to changes in binding affinity to C1q by aglycosylation,IgG1 Fc having the strongest complement activity markedly decreased whenaglycosylated. IgG4 Fc, known not to induce complement activation,rarely had binding affinity to C1q, indicating that the IgG4 Fc is usedas an excellent recombinant carrier with no complement activity (FIG.17).

To determine whether the carrier maintains its property of having nobinding affinity to C1q even after being conjugated to a physiologicallyactive peptide, IFN alpha-Fc conjugates were prepared using glycosylatedFc, enzymatically deglycosylated Fc and aglycosylated recombinant Fc ascarriers for IFN alpha and were evaluated for their binding affinity toC1q. A glycosylated Fc-coupled IFN alpha conjugate (IFNα-PEG-Fc:Glycosylated IgG1Fc) maintained a high binding affinity to C1q. Incontrast, when interferon alpha was coupled to an Fc deglycosylatedusing PNGase F and other enzymes, the resulting conjugate(IFNα-PEG-DGFc: Deglycosylated IgG1Fc) displayed a markedly decreasedbinding affinity to C1q, which was similar to that of the E.coli-derived aglycosylated Fc conjugate. In addition, when the IgG1moiety of the aglycosylated IgG1 Fc-coupled interferon alpha conjugate(IFNα-PEG-AGFcG1: Aglycosylated IgG1Fc) was exchanged with the IgG4moiety, the resulting interferon conjugate (IFNα-PEG-FcG4 derivative 1:Aglycosylated IgG4Fc) was found to completely lose its binding affinityto C1q. When the IgG1 Fc moiety was exchanged with the IgG4 Fc monomer,the resulting conjugate (IFNα-PEG-FcG4 derivative 2: AglycosylatedIgG4Fc). These results indicate that such forms of the IgG4 Fc fragmentare useful as excellent carriers not having the effector functions ofantibody fragments (FIG. 18).

INDUSTRIAL APPLICABILITY

As described hereinbefore, the IgG Fc fragments of the present inventionincrease serum half-lives of drugs and sustain in vivo activity of drugswhen used as carriers. In particular, the present IgG Fc fragmentsincrease serum half-lives of polypeptide drugs to levels higher than anyconventional modified proteins, and overcome the most significantdisadvantage of conventional long-acting formulations, decreased titers,thus having blood circulation time and in vivo activity superior toalbumin, previously known to be most effective. In addition, the presentIgG Fc fragments have no risk of inducing immune responses. Due to theseadvantages, the present IgG Fc fragments are useful for developinglong-acting formulations of protein drugs. Further, the long-actingformulations of protein drugs according to the present invention arecapable of reducing the patient's pain from frequent injections, andmaintaining serum concentrations of active polypeptides for a prolongedperiod of time, thus stably providing pharmaceutical efficacy.

What is claimed is:
 1. An isolated polynucleotide encoding an IgG Fcfragment as a drug carrier, wherein the IgG Fc fragment is aglycosylatedand consists of the amino acid sequence of SEQ ID NO:
 10. 2. Thepolynucleotide as set forth in claim 1, which consists of the nucleotidesequence of SEQ ID NO:
 9. 3. A recombinant vector comprising thepolynucleotide of claim
 2. 4. A transformant transformed with therecombinant vector of claim
 3. 5. A method of preparing an Fc fragment,comprising culturing the transformant of claim 4 in a medium under acondition allowing the transformant to express the Fc fragment andisolating the Fc fragment.